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What is Corallina officinalis var. chilensis? An examination of nomenclature, biogeography, phylogeny,… Huber, Soren 2020

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 What is Corallina officinalis var. chilensis? An examination of nomenclature, biogeography, phylogeny, and morphology  by   Soren Huber B.M., University of Maryland, College Park, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September 2020  © Soren Huber, 2020  ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled:  What is Corallina officinalis var. chilensis?  An examination of nomenclature, biogeography, phylogeny, and morphology  submitted by Soren Huber  in partial fulfillment of the requirements for the degree of Master of Science in Botany  Examining Committee: Patrick Martone, Professor, Department of Botany, UBC Supervisor  Amy Angert, Associate Professor, Department of Botany, UBC Supervisory Committee Member  Mary Berbee, Professor, Department of Botany, UBC Supervisory Committee Member Quentin Cronk, Professor, Department of Botany, UBC Additional Examiner     Additional Supervisory Committee Members: Paul Gabrielson, Adjunct Assistant Professor, Department of Biology, UNC  Supervisory Committee Member         iii Abstract    Geniculate coralline algae are notoriously challenging to identify in the field due to confusing morphological variation. Consequently, former species delimitations based exclusively on morphology are often unsupported by sequence-based phylogenies. The purpose of my research was to determine whether Corallina chilensis Decaisne, basonym of C. officinalis var. chilensis, was a distinct species or should be considered a variety of C. officinalis; and consequently whether C. chilensis was distributed in two hemispheres.   In order to answer these questions, I sequenced psbA, CO1, and rbcL genes from 76 voucher specimens representing Corallina collections from ~2000 to 2019. I applied names by comparing these sequences with published sequences and type specimen sequences, including an rbcL sequence from the specimen collected by Darwin (#2151 from Valparaiso, Chile), the holotype specimen for C. chilensis designated by Harvey. I used phylogeny with additional support from morphometric, Automatic Barcode Gap Discovery, and distance matrix analyses for species delimitation.   DNA from the Chilean C. chilensis holotype matched an unnamed coralline species commonly found in the Northeast Pacific, and C. chilensis specimens formed a separate clade from C. officinalis specimens in my phylogenetic analyses. Corallina chilensis is a distinct species, not a variety of C. officinalis, and it is present in both hemispheres. Going forward, the name C. officinalis var. chilensis should be discontinued, and the older name C. chilensis should be used in its place.        iv Lay Summary   Algae come in many different colors, shapes, and sizes, and there are hundreds of species present in oceans worldwide. Sometimes it is hard to tell two species apart because they look so similar, while other times one species can have many different appearances. Thus, we must use DNA sequence data to confirm species identity and to ensure each species is given its proper binomial name.   This research involved extracting and sequencing DNA from a specimen collected by Charles Darwin, during a stop by the HMS Beagle at Valparaiso, Chile, and comparing it to our recent collections. We discovered that Darwin’s original specimen corresponded with a species growing in the Pacific Northwest, and that this species was not a variety of Corallina officinalis. Given that it is a distinct species, it should not be called “C. officinalis var. chilensis,” but rather C. chilensis, the name originally applied to Darwin’s Chilean specimen.                       v Preface   I wrote this thesis based on work conducted in the Martone Laboratory (Botany Department, University of British Columbia), with collaborators Jeffery Hughey and Paul Gabrielson. I was responsible for molecular work in the laboratory, for the phylogenetic analyses, and for the overseeing and implementing of morphometric measurements.   Katherine Hind, Patrick Martone, and Paul Gabrielson were the primary collectors of specimens used in this research. Erasmo Macaya sent two specimens from Chile. Jeffery Hughey and Paul Gabrielson extracted and amplified DNA from the three 1800’s herbarium specimens. Paul Gabrielson also contributed a sequence of Corallina chilensis from Chile. Jasmine Lai sequenced PTM specimens 1984 and 1985. Some DNA sequences, particularly the psbAF1 DNA sequences used for identification, were retrieved from the laboratory archive that had been sequenced by Katherine Hind or former lab volunteers and work-study students.  Brenton Twist and Mary Berbee provided advice on statistical analyses for phylogenetic trees and aided in interpreting the results. Jade Shivak took all morphometric measurements (in Table 4 and Appendix III); created the map in Figure S12, Appendix II; took the photographs that appear in Figure 25; and prepared R graphs in Figure 26. Patrick Martone took the photograph in Figure 27, and Bill Woelkerling took the photographs in Figures 4 and 5. Modifications to the figures and photographs mentioned above and all other figures and photographs are my own.  Patrick Martone, Mary Berbee, Paul Gabrielson, and Amy Angert contributed manuscript edits.     vi Table of Contents  Abstract ..................................................................................................................................... ii Lay Summary .......................................................................................................................... iv Preface....................................................................................................................................... v Table of Contents ..................................................................................................................... vi List of Tables ........................................................................................................................... xi List of Figures ......................................................................................................................... xii List of Symbols and Abbreviations ....................................................................................... xiv Acknowlegements ................................................................................................................... xv Dedication ............................................................................................................................. xvii Introduction .............................................................................................................................. 1 I. Corallines and their significance .......................................................................................... 2 II. Identification and species delimitations .............................................................................. 4 III. Nomenclature and the importance of sequencing type specimens ...................................... 7 IV. Corallina officinalis var. chilensis .................................................................................... 9     vii Chapter I: What is Corallina officinalis var. chilensis? ........................................................ 12 1.1 Introduction .................................................................................................................... 12 1.1.1 Historical context ............................................................................................. 12 1.1.2 Study objectives ............................................................................................... 20 1.2 Materials and methods .................................................................................................... 21 1.2.1 Sampling ......................................................................................................... 21 1.2.2 DNA extraction, amplification, and sequence assembly ................................... 22 1.2.3 Sequence alignment & phylogenetic analysis ................................................... 25 1.2.4 Other analyses supporting species delimitation ................................................ 27 1.2.5 Morphometric analysis..................................................................................... 28 1.3 Results ............................................................................................................................ 31 1.3.1 Corallina chilensis is not a variety of C. officinalis .......................................... 48 1.3.2 Specimens from the Northern Hemisphere matched the C. chilensis holotype & formed a clade in all trees. ........................................................................................ 50 1.3.3 Other specimens thought to have been C. chilensis based on morphology ........ 51 1.3.4 Analysis of conflict and congruence among gene trees ..................................... 52 1.3.5 Distribution of C. chilensis. ............................................................................. 55   viii 1.3.6 Morphological measurements .......................................................................... 57 1.3.7 Morphological description of C. chilensis in the Northeast Pacific ................... 58 1.4 Discussion ...................................................................................................................... 64 1.4.1 Identity and rank of C. chilensis ....................................................................... 64 1.4.2 Known global distribution of Corallina, and specifically C. chilensis .............. 66 1.4.3 How to identify C. chilensis in British Columbia, Canada ................................ 69 1.4.4 Phylogenetic position of C. chilensis within Corallina ..................................... 71 1.4.5 Incongruence across coralline gene trees: an anomaly or more common than we think? ....................................................................................................................... 73 1.4.6 Conclusions ..................................................................................................... 76 1.4.7 Future directions with respect to C. chilensis ................................................... 76 Chapter II: Future Directions in Corallina ............................................................................ 78 2.1 Introduction .................................................................................................................... 78 2.2 Examination of currently accepted Corallina species ...................................................... 79 2.2.1 Evaluation of the generitype C. officinalis Linnaeus......................................... 79 2.2.2 Evaluation of accepted species C. vancouveriensis Yendo ............................... 79 2.2.3 Evaluation of accepted species C. crassissima, C. declinata, and C. aberrans .. 80   ix 2.2.4 Evaluation of accepted species C. ferreyrae ..................................................... 81 2.2.5 Evaluation of accepted species C. maxima ....................................................... 82              2.2.6 Evaluation of accepted species C. melobesioides ............................................. 83 2.2.7 Evaluation of accepted species C. pinnatifolia ................................................. 83 2.2.8 Summary of evidence supporting or rejecting currently accepted species designations.............................................................................................................. 84 2.3 Provisionally identified Corallina species ....................................................................... 84 2.3.1 C. sp. 3 frondescens & C. sp. 3 frondescens-like .............................................. 84 2.3.2 C. sp. 1 gws & C. sp. 1 gws-like ...................................................................... 86 2.3.3 C. sp. 2 vancouveriensis ................................................................................... 87 2.3.4 Taxa surrounding C. ferreyrae ......................................................................... 88 2.3.5 C. sp. 2 chile complex within the C. ferreyrae clade ........................................ 90 2.3.6 C. sp. 1 california ............................................................................................. 91 2.3.7 C. sp. 4 frondescens ......................................................................................... 92 2.3.8 C. sp. 2 frondescens ......................................................................................... 92 2.3.9 C. sp. 5 frondescens ......................................................................................... 93 2.4 Conclusion ...................................................................................................................... 94   x References ............................................................................................................................... 97 Appendices ............................................................................................................................ 109 Appendix I: Historical materials ......................................................................................... 109 Appendix II: Sequences ...................................................................................................... 119 Appendix III: Morphological measurements ....................................................................... 126 Appendix IV: Methods tables ............................................................................................. 128 Appendix V: Distance matrices ........................................................................................... 129             xi List of Tables Table 1. Table of primer names, sequences, and primer sources ............................................... 23 Table 2. ABGD barcoding analyses performed on psbA, CO1, and rbcL.................................. 32 Table 3. Summary table of C. chilensis percent differences ...................................................... 50 Table 4. Summary table of morphological measurements ......................................................... 58 Table 5. Percent differences across C. crassissima complex ..................................................... 81 Table 6. Percent differences across the C. sp. 3 frondescens complex ....................................... 86 Table 7. Percent differences across the C. sp. 1 gws complex ................................................... 87 Table 8. Percent differences across the C. vancouveriensis complex ........................................ 88 Table 9. Percent differences across the C. ferreyrae complex ................................................... 91 Table 10. Percent differences between other provisionally named Corallina species ................ 94 Table S1. Table of all sequence data ...................................................................................... 119 Table S2. Concatenated outgroup sequences .......................................................................... 123 Table S3. Table of C. chilensis specimens collected by Hind & Saunders (2013A) ................ 123 Table S4. Corallina chilensis measurements for morphological analysis ................................ 126 Table S5. Corallina vancouveriensis measurements for morphological analysis ..................... 126 Table S6. Thermal cycler settings .......................................................................................... 128 Table S7. Rates of evolution and models for tree analyses ...................................................... 128 Table S8. Percent difference matrix of psbA sequences .......................................................... 129 Table S9. Percent difference matrix of CO1 sequences........................................................... 130 Table S10. Percent difference matrix of rbcL sequences. ....................................................... 131    xii List of Figures  Figure 1. Corallines growing in situ and diagram of coralline frond ........................................... 2 Figure 2. Morphological variation in C. vancouveriensis............................................................ 5 Figure 3. Similar morphology exhibited by different coralline species ....................................... 5 Figure 4. Darwin’s C. chilensis specimen from Valparaiso, Chile ............................................ 13 Figure 5. Corallina sp. collected by Gay from Ancud, Chile in 1836 ....................................... 15 Figure 6. Kützing’s 1858 description of C. officinalis [var.] chilensis ...................................... 17 Figure 7. Summary of measurements taken for morphometric analysis .................................... 30 Figure 8. rbcL gene tree including sequence from C. chilensis type specimen .......................... 33 Figure 9. Expanded portion of the rbcL tree from Fig. 8. ......................................................... 34 Figure 10. psbA gene tree ........................................................................................................ 35 Figure 11. Expanded portion of psbA tree from Fig. 10 ............................................................ 36 Figure 12. CO1 gene tree ......................................................................................................... 37 Figure 13. Expanded portion of the CO1 tree from Fig. 12 ....................................................... 38 Figure 14. rbcL gene tree excluding C. chilensis type & short sequences ................................. 39 Figure 15. Expanded portion of the rbcL tree from Fig. 14. ...................................................... 40 Figure 16. Majority rule tree .................................................................................................... 41 Figure 17. Majority rule tree expanded from Fig. 16 ................................................................ 42 Figure 18. Majority rule tree expanded from Fig. 16 ................................................................ 43 Figure 19. Majority rule tree expanded from Fig. 16. ............................................................... 44 Figure 20. Concatenated tree .................................................................................................... 45   xiii Figure 21. Concatenated tree expanded from Fig. 20 ................................................................ 46 Figure 22. Concatenated tree expanded from Fig. 20 ................................................................ 47 Figure 23. Map of C. chilensis' recently confirmed range using DNA ...................................... 56 Figure 24. Morphologically variable C. chilensis collections from British Columbia ................ 60 Figure 25. Side-by-side comparison of C. chilensis and C. vancouveriensis ............................. 61 Figure 26. Intergenicular dimensions of C. chilensis and C. vancouveriensis ........................... 62 Figure 27. In situ photograph of C. chilensis. ........................................................................... 63 Figure S1. Linnaeus' (1758) description of C. officinalis ........................................................ 109 Figure S2. Ellis' (1755) illlustration of C. officinalis .............................................................. 110 Figure S3. Schmitz' (1889) designation of Corallina genus .................................................... 111 Figure S4. Harvey's (1849) Nereis Australis publication of C. chilensis ................................. 112 Figure S5. Montagne's (1852) report of C. chilensis ............................................................... 113 Figure S6. Ardissone's (1888) report of C. officinalis var. chilensis........................................ 114 Figure S7. Yendo's (1902A) report of C. officinalis var. chilensis .......................................... 115 Figure S8. Setchell's (1903) report of C. officinalis var. chilensis ........................................... 116 Figure S9. Foslie's (1907) report of C. chilensis ..................................................................... 116 Figure S10. Skottsberg's (1923) report of C. chilensis ............................................................ 117 Figure S11. Smith's (1944) report of C. chilensis ................................................................... 118 Figure S12. Map of C. chilensis sampling sites north of Oregon, USA ................................... 125         xiv List of Symbols and Abbreviations    ABGD – Automatic Barcode Gap Discovery aLRT – approximate Likelihood Ratio Test bp – base pair bs – bootstrap CO1 – Cytochrome c oxidase subunit I ML – Maximum Likelihood PC – Paris herbarium  PCR – Polymerase chain reaction PTM – Patrick T. Martone (prefix designation for specimens collected by Martone Laboratory) psbA – Photosystem II protein D1 precursor rbcL – Ribulose bisphosphate carboxylase large subunit TCD – Trinity College Herbarium UBC – University of British Columbia            xv Acknowledgements  I would first like to acknowledge my thesis advisor, Patrick Martone, for accepting the risk and allowing a music major into his lab. I’ve always appreciated Patrick’s enthusiasm, love of seaweeds, and fountain of ideas. Most of all though, I have admired the tone he established and the culture of care, respect, and inclusivity that he cultivated in his lab. The Martone lab has been a second family to me the past 3 years. My lab mates taught me the ropes, including how to actually “do research” in practice, aided me with field work and experiments, critiqued my drafts and presentations, and were there for me through the best and worst of times. I’ll never forget the fun times we had together “cooking” in the lab, on collecting trips, attending conferences and writing retreats, at curling championships, and playing “salad bowl.”  During this degree program, I have been very fortunate to have had an exceptional Supervisory Committee. Thanks to Paul Gabrielson for entrusting me with this special project and for having patiently answered dozens of questions and helping me get all the wording just right. I am appreciative to Amy Angert for her perspective, direction, and advice. An especially huge thank you goes to Mary Berbee, (AKA “Mary Godmother”), for helping me troubleshoot PCR when all else failed, interpreting unexpected results, suggesting cool additional analyses and walking me through how to execute them, and contributing hours of edits so this research might be adequately communicated to the rest of the world. I thank Mary for her ever-optimistic perspective, commitment to good science despite the extra time investment, help in framing this story, and encouragement to produce the best possible product.   I would like to recognize Jade Shivak for her strong work ethic, upbeat attitude, and complete trustworthiness while measuring hundreds of coralline intergenicula, among other things, under the microscope and graphing those data for this thesis.   xvi Many thanks to my students from Biol 209, 230, and 320. They were the reason I jumped out of bed for an early start during long rainy winters, and they taught me how to teach. Working with them was a highlight and one of the most rewarding aspects of grad school.    It has certainly taken a village to raise this botanist, and I would like to acknowledge the following people for their support during this journey. Lisa Brooks has been my long-term unofficial biology mentor and compass who pointed me in the right direction from the beginning. Sandy Wyllie-Echeverria gave me a critical foothold when I needed prerequisite research experience. Jeff Hughey was instrumental in helping me access the best possible place to study exactly what I wanted to study. Although he is no longer with us, I would like to take this opportunity to acknowledge Len Dyck, to whom this thesis is dedicated. Both artist and scientist, Len loved his research and teaching, was humble despite his brilliance, and exceedingly kind to his students even bringing them cookies to stave off “the shakes” during lab exams. Len, thank you for all that you taught me about algae, for the perspective you offered me on my thesis research, but mostly for the example you set as an instructor and human being. I am so lucky to have had the opportunity to work with you. You will always be remembered.  Finally, I would like to acknowledge my entire family for their unconditional love and support. Special thanks to my father, Carl Huber, and my grandmother, Jeanne Huber, for sharing their love of plants with me for as long as I can remember; to my grandfather, Al Huber, for lessons in the garden and the fields; and to my mother, Leah Huber, for encouraging me to collect, press, and study plants and fungi from an early age. I am grateful to Ross Schipper for his overall support and infinite optimism, including through a pandemic; but most of all for celebrating all the failures and successes with me.    xvii Dedicated in memory of  Len Dyck   (Who understood that there is more to “species” than “just a few base pairs.”)       1 Introduction    Marine macroalgae, or “seaweeds,” are members of a morphologically diverse group of photosynthetic eukaryotes that inhabit all the planet’s oceans. Ranging in size from hardly visible filamentous strands to 50-meter-long foliose kelps (Graham et al. 2009), seaweeds display a vast pallet of colors, textures, sizes, and shapes for adapting to hydrodynamic, temperature, and, for some, desiccation stress characteristic of coastal habitats (Armstrong 1989, Blanchette et al. 2002, Boller & Carrington 2006, Collado-Vides 2002, Monro & Poore 2005, Koehl et al. 2008). Macroalgae can be red, green, brown, pink, purple, yellow, or black, depending on the combination of specialized pigments they contain for photoprotection, and for absorbing light underwater (Graham et al. 2009).    Seaweed size and shape is highly variable yielding many different morphologies, i.e. ways that seaweeds can look. Having a highly variable morphology can be confusing if one is trying to identify seaweeds to species and has been the subject of phycological study for a long time. Many seaweeds can look similar due to heredity, but in other cases, morphology depends upon the conditions in which they are growing (Ramus 1972, Denny et al. 1985, Armstrong 1988, Armstrong 1989, Gaylord et al. 1994, Blanchette 1997, Blanchette et al. 2002, Collado-Vides 2002, Monro & Poore 2005, Boller & Carrington 2006). For instance, in high flow environments some kelp blades tend to be narrow and flat, while in slower flow environments they tend to be wider and ruffled (Gerard & Mann 1979, Armstrong 1988, Armstrong 1989). Higher in the intertidal zone where the shore is exposed to air for hours at a time, other algae have adapted to desiccation by reducing their surface to volume ratio or growing as turfs to maximize water retention while the tide is out (Padilla 1984, Hunt & Denny 2008, Holzinger & Karsten 2013, Guenther & Martone 2014). Thus, turf forming algae tend to be small (millimeters   2 to only a few centimeters), highly branched, and frilly (Gaylord et al. 1994). We do not understand the morphological variation exhibited by most seaweed species, and this has been (and continues to be) problematic for taxonomists and other researchers over the years.    I. Corallines and their significance    Coralline red algae (Phylum Rhodophyta, Subphylum Eurhodophytina) are situated within the class Florideophyceae. Members of Florideophyceae are typically characterized by a triphasic life history (Graham et al. 2009). The common name “coralline” specifically refers to three orders, Corallinales, Hapalidiales and Sporolithales, within the subclass Corallinophycidae (Hind et al. 2018). This group is characterized by their ability to incorporate calcium carbonate into their cell walls, mostly in the form of high-magnesium calcite (Hippler et al. 2009, Smith et al. 2012, Nash et al. 2017). Calcium-carbonate impregnated cell walls give corallines a hard, rock-like quality, and chalky, pink-purple appearance (Fig. 1).   Figure 1. (A) Corallines growing on rock under kelp. (B) Articulated coralline joint “geniculum.” (C) Coralline fronds growing in tidepool. (D) Diagram of generic coralline frond, arrows pointing to intergenicula.  A BCcrownstemD  3 Corallines have a range of forms. Some occur as crusts completely adhering to the substratum, others occur as free-living rhodoliths unattached to any substrata and ranging in size from pebbles to small boulders. Still other corallines grow as upright, segmented “articulated” fronds (Fig. 1A-D) several centimeters high (Johansen 1981, Gabrielson & Lindstrom 2018). While there are exceptions to these generalizations, for the purposes of clarity, in this thesis I will refer to encrusting and rhodolith-like morphologies as “non-geniculate corallines,” and upright articulated forms as “geniculate corallines.” All coralline individuals begin from a single spore that divides to form a basal crust. Geniculate corallines grow upright from their basal crusts and tend to have a lower portion of unbranched axes that divide to form clusters of branches, which I call “crowns” (Fig. 1D). Fronds may grow individually (Fig. 1C) or in clumps (Fig. 1B), and can exhibit a variety of branching patterns including pinnate, irregular, dichotomous, planar, or whorled. Geniculate corallines are composed of many hard, calcified, longer segments separated by soft, very short, uncalcified regions that act as joints (Fig. 1B-D). The joints between segments are referred to as “genicula,” (Fig. 1B), and the calcified segments between genicula are called “intergenicula” (Fig. 1D). Joints lend fronds the ability to flex and bend. This enables geniculate corallines to live in high-energy wave swept environments and thrive where few other organisms can survive (Johansen 1981, Martone 2006, 2007, Martone & Denny 2008A, 2008B, Denny et al. 2013, Janot & Martone 2016). Genicula have evolved at least three different times throughout evolutionary history, which is reflected in their distribution in three different subfamilies Metagoniolithoideae, Lithophylloideae, and Corallinoideae (Janot & Martone 2018). Size and shape of intergenicula may sometimes be used to help differentiate among species or genera in the field (Abbott & Hollenberg 1976, Johansen 1981, Baba et al. 1988), but intergenicular   4 morphology is notoriously problematic and may not consistently be used as a diagnostic character (see Hind et al. 2014A, 2014B).   II. Identification and species delimitations    While non-geniculate coralline algae have a long history of being challenging to identify (Sissini et al. 2014, van der Merwe et al. 2015, Maneveldt et al. 2017, Twist et al. 2019), it turns out that articulated corallines may be just as challenging to identify. Therefore field identifications of geniculate corallines must also be confirmed or rejected by comparing DNA sequences of unknown specimens with DNA sequences from specimens whose identities have been established.  Corallines are challenging to identify because a single species can be so morphologically variable that specimens of the same species can appear to be multiple species. For example, individuals of Corallina vancouveriensis growing only a few meters apart can appear morphologically different from one another (Fig. 2). Corallina vancouveriensis specimens may grow as brush-shaped fronds (Fig. 2A) with irregular branches and pinnules (i.e. small secondary terminal branchlets), may grow as flat, symmetrical fronds (Fig. 2B), or both flat and brush-shaped fronds from the same basal crust (Fig. 2C). In other instances, geniculate coralline field identifications may be confounded because multiple coralline species appear morphologically similar and may be mistaken for the same species (Fig 3). For example, some species of Corallina and Bossiella can look remarkably similar in the field (Fig. 3).    5  Figure 2. Corallina vancouveriensis growing on North Beach, Calvert Island, BC, Canada. (A) Exposed brush like form. (B) Shaded flat form. (C) Both forms in one clump. Arrow is pointing to flat fronds towards the middle of the clump where they are shaded by the outer brush-shaped fronds.     Figure 3. Corallines growing at Botanical Beach, British Columbia, Canada. (A) Bossiella sp. (B) Corallina sp.   Several different phenomena have led to taxonomic confusion in the corallines. These include convergent traits between distant relatives, that is, similar traits that have evolved independently multiple times (Janot & Martone 2018); nearly identical morphology between closely related species (i.e. cryptic speciation) (Gabrielson et al. 2011, Brodie et al. 2013, Sissini A B CA B  6 et al. 2014, Hind et al. 2015); and morphological variation between close relatives, or “intraspecific variation,” sometimes based on habitat or geographic location (Hind et al. 2015 Hind et al. 2014B, Hind et al. 2016, Hind et al. 2018, Jeong et al. 2019). Also, some corallines appear to change their morphology based on environmental influences, i.e. they exhibit phenotypic plasticity (Tyrell & Johansen 1995, DeWitt & Scheiner 2004, Maneveldt & Keats 2008). These phenomena in isolation or combination have led to instances where one name has been applied to multiple species (Gabrielson et al. 2011, Hind & Saunders 2013A, Hind et al. 2014B, Sissini et al. 2014, Hind et al. 2015) and other cases where multiple names were applied to the same species or genus (Hind et al. 2014A, van der Merwe et al. 2015, Hind et al. 2016, Hind et al. 2018, Jeong et al. 2019). Historically, coralline taxonomy was based exclusively on morpho-anatomy and, consequently, due to the above-mentioned phenomena, names were frequently misapplied. Current studies implement DNA sequence data to designate species boundaries (Leliart et al. 2014, van der Merwe et al. 2015, Nelson et al. 2015, Hind et al. 2016, Spalding et al. 2016, Richards et al. 2017, Hind et al. 2018, Twist et al. 2019), and subsequently to determine distinguishing morphological characteristics, if any exist, based on those genetic boundaries.    The process of reconciling old and new approaches of identification and species delimitation has led to vast taxonomic fluctuation (Gabrielson et al. 2011, Brodie et al. 2013, Hind & Saunders 2013A, Hind & Saunders 2013B, Hind et al. 2014A, Hind et al. 2015, Hind et al. 2016, Rösler et al. 2016, Bustamante et al. 2019). As a result, there is an abundance of putative coralline species with provisional names in the literature that require confirmation and description (Saunders & Hommersand 2004, Le Gall et al. 2010, Martone et al. 2012, Hind & Saunders 2013A, Hind et al. 2016, Yang et al. 2016). Reconciling old and new approaches and   7 confirming and describing putative coralline species is necessary to obtain accurate biodiversity estimates (Kucera & Saunders 2012, Brodie et al. 2013, Williamson et al. 2015).  A species definition common across multiple species concepts is helpful when reconciling morpho-anatomical based approaches and DNA sequence-based approaches to species delimitation. Species may thus be defined as “separately evolving metapopulations” (De Queiroz 2007). Evidence that metapopulations are evolving separately may include reproductive or geographical isolation, as well as morphological, molecular, or phylogenetic distinction (De Queiroz 2007). Congruence across multiple lines of evidence is advisable for delimiting species (Carstens et al. 2013). Some molecular-based techniques implemented in species delimitation of algae include use of phylogenetic trees, Automatic Barcode Gap Discovery (ABGD) analyses, and the comparison of DNA sequences in distance matrices (Le Gall & Saunders 2010, Hind & Saunders 2013A, Nelson et al. 2015, van der Merwe et al. 2015, Jeong et al. 2019, Twist et al. 2019). In my research, I used a combination of aLRT (approximate Likelihood Ratio Test), Bayesian, and bootstrap support for monophyly in both individual and concatenated gene sequences to delimit species, with the consistent separation of species in ABGD analyses as further confirmation of speciation. I also looked for morphological differences between my study species and a congeneric species that is commonly found growing in the same vicinity in Northeast Pacific populations.  III. Nomenclature and the importance of sequencing type specimens   In botanical nomenclature, that includes vascular and non-vascular plants, algae, and fungi, each published name is permanently attached to an original “type” specimen, to which all other specimens can be compared. To clearly understand the species to which a name is   8 referring, researchers must link that name to the original type collection (Turland et al. 2018, see article 7.2). With respect to red algae, this was accomplished until 2001 using morpho-anatomy, but is ideally done by extracting DNA from type specimens for comparison with the DNA sequences from specimens in question. Many type specimens were collected in the 1700’s and 1800’s, and specific primers and protocols are required to extract remaining intact fragments of partially degraded DNA (Hughey et al. 2001, 2002, Gabrielson et al. 2011). Hughey et al. (2001, 2002) were the first researchers to successfully extract and amplify DNA from red algal type specimens in the family Gigartinaceae for the purposes of molecular comparison. Gabrielson et al. (2011) adapted the technique to geniculate coralline algae where it has been used to correctly apply names in the geniculate genera Calliarthron, Corallina (Hind et al. 2014A), and Bossiella (Hind et al. 2014B, 2015).  This approach of using DNA sequences from type materials for comparison with recent collections enabled researchers to determine that species formerly thought to have been Calliarthron belonged to a different genus, that three species were synonymous, and that there were only two Calliarthron species (Gabrielson et al. 2011). Sequencing type material in another study demonstrated that what was formerly referred to as Pachyarthron cretaceum based on morphology, was molecularly identical to and should be called Corallina officinalis (Hind et al. 2014A). In the case of Bossiella, which was originally thought to consist of fewer species because of overlapping morphological characters across multiple species, DNA sequences from type specimens were correlated with genetic groups to recognize and describe over a dozen species within the genus (Hind et al. 2015, Hind et al. 2018).  While DNA sequencing of old type material has been successfully incorporated into many red algal taxonomic studies over the past two decades, the practice has not been   9 implemented consistently across the field (Farr et al. 2009, Walker et al. 2009, Nelson et al. 2015, Melbourne et al. 2017). Any given collection of DNA sequences may be compared and divided into molecular species groups or compared to DNA sequences published in databases or other publications, yet not comparing such groups with types and thus anchoring them to original names, can create confusion (Walker 2009, Bustamante 2019).  In some cases, it is not possible to utilize type DNA because the type specimen could not be located (e.g. Yendo’s Corallina collections have not been found), or DNA could not be successfully extracted and amplified from old type material (Brodie et al. 2013). Resolving the nomenclature may still be possible. For instance, Brodie et al. (2013) selected an epitype for Corallina officinalis when they could not successfully extract and amplify DNA from the designated lectotype (BM 001062598).  In other cases, morpho-anatomical features are still used to compare specimens with types. Nelson et al. (2015) compared their specimens to Harvey et al. (2005) specimens which had been identified based on morph-anatomical examination of type specimens.  IV. Corallina officinalis var. chilensis    The subject of this thesis, Corallina officinalis var. chilensis (Decaisne) Kützing (1858), is a perfect example of the challenges facing the identification, delimitation and naming of coralline algal species. Corallina officinalis var. chilensis is a geniculate coralline belonging to the order Corallinales (Silva & Johansen 1986), and member of the family Corallinaceae (Lamouroux, 1812), characterized by grouped, zonate-divided, tetra- and bi-sporangia that have no plugs and are housed in uniporate, calcified conceptacles (Harvey et al. 2003). Within Corallinaceae, there are currently seven recognized subfamilies; Lithophylloideae (Setchell   10 1943), Corallinoideae (Areschoug) Foslie 1908, Chamberlainoideae (Caragnano, Foetisch, Maneveldt & Payri 2018), Neogoniolithoideae (Kato & Baba 2011), Mastophoroideae (Setchell 1943), Metagoniolithoideae (Johansen 1969), and Hydrolithoideae (Kato & Baba 2011). Corallina is one of 13 recognized genera in the subfamily Corallinoideae (Hind & Saunders 2013A, Hind et al. 2016, Hind et al. 2018, Guiry & Guiry 2020). Corallina contains nearly as many provisionally named species as species that have been formally described and are supported by a morpho-anatomical comparison or DNA sequence match to their type specimen (Hind and Saunders 2013A). Supported species to date include C. aberrans (Yendo) K.R.Hind & G.W.Saunders, C. declinata (Yendo) K.R.Hind & G.W.Saunders, C. crassissima (Yendo) K.R.Hind & G.W.Saunders 2013, C. officinalis Linnaeus, C. maxima (Yendo) K.R.Hind & G.W.Saunders, C. vancouveriensis Yendo, C. ferreyrae E.Y.Dawson, O.C. Acleto, & N. Foldvik, C. pinnatifolia (Manza) E.Y.Dawson, and C. melobesioides (Segawa) P.T.Martone, S.C.Lindstrom, K.A.Miller, & P.W.Gabrielson 2012. Putative species in need of confirmation in addition to C. officinalis var. chilensis include C. sp. 2 frondescens, C. sp. 3 frondescens, C. sp. 4 frondescens, C. sp. 5 frondescens, C. sp. 1 gws, C. sp. 2 vancouveriensis, C. sp. 1 california, and C. sp. 5 korea (Hind & Saunders 2013A). Typical of many corallines, Corallina species are difficult to tell apart in the field due to cryptic speciation and/or variable morphology. Of the Corallina species in the Northeast Pacific, C. vancouveriensis appears to be the most common inhabitant of rocky intertidal zones, although it may be sometimes challenging to identify in the field. Other Corallina species are found less frequently and are also difficult to differentiate based on morphology. While the name “C. officinalis var. chilensis” has been haphazardly applied for decades without type consultation, this is the first time that DNA has been extracted and sequenced from its holotype specimen to determine the accurate application of its name.   11   In this thesis, I first investigate which specimens from our recent collections are indeed C. officinalis var. chilensis by comparing DNA sequences from recent (e.g., collected after ~ year 2000) collections with a DNA sequence from the holotype specimen. Then I describe the species within the context of its genus, update its distribution based on sequenced specimens, and characterize Northeast Pacific populations based on morpho-anatomy.             12 Chapter I What is Corallina officinalis var. chilensis?    1.1 Introduction  1.1.1 Historical context  Linnaeus (1758), when he proposed Corallina, listed the binomial names and descriptions of 10 species, including Corallina officinalis, but he did not designate a generitype species (Appendix I, Fig. S1). In the original description of C. officinalis, Linnaeus referenced an illustration by Ellis (1755, Appendix I, Fig. S2), which by definition was considered the holotype (Turland et al. 2018). The type locality for C. officinalis was “Habitat in Oceane Europaeo, Americano” (Linnaeus 1758). Schmitz (1889) placed Corallina within the family Corallinaceae situated within the “Florideen” [subclass Florideophycidae], and designated C. officinalis as the generitype (Appendix I, Fig. S3). Irvine in Jarvis (1993: 37) designated a specimen in the Linnaean Herbarium LINN 1293.9 as the lectotype. Recently, Brodie et al. (2013) were unable to obtain any viable DNA sequences from the lectotype specimen (LINN 1293.9 from Linnaeus’ collection) and designated a neotype specimen from which DNA was successfully extracted and amplified (Spencer et al. 2009, Brodie et al. 2013).   Nearly a century after Linnaeus described Corallina officinalis from the Northern Hemisphere, Irish botanist and phycologist William Henry Harvey (1849) published in Nereis Australis descriptions of coralline algal taxa in the southern oceans including C. chilensis Decaisne in Harvey (Appendix I, Fig. S4). The holotype of C. chilensis that is cited in the   13 description is "Valparaiso C. Darwin 2151," a collection made by Charles Darwin that is currently housed in Trinity College Herbarium (TCD) (Fig. 4).   Figure 4. Darwin’s C. chilensis specimen from Valparaiso, Chile. Housed at the  Trinity College Herbarium, Dublin, Ireland (Appendix II). This specimen was  designated the type specimen in Harvey 1849.  Photos:  Bill Woelkerling  14 Harvey (1849) reported that C. chilensis was also collected from Port Famine (C. Darwin #1840) and from Norfolk Island [Australia]. There was no collection number provided in the description for the Norfolk Island specimen, but Harvey reported that the collection resides in “Herb. Hooker” (see Appendix I, Fig. S4). The description was as follows:  “1-2 inches high, bi-tri-pinnate above, the pinnae long, erecto-patent, the upper ones  gradually shorter. Articulations of the stem and branches once and half as long as broad,  cuneate, simple, the upper ones longer and more expanded towards the apex, very  irregular in shape, often laciniate or crenate; the apical ones, especially, frequently  palmate” (Harvey, 1849). Note that Harvey credited the French Belgian botanist Joseph Decaisne in “Herb. Paris” for the description (Harvey, 1849). Decaisne, who was at the Muséum National d’Histoire Naturelle, may have seen other C. chilensis specimens in the Paris herbarium (PC), but may never have seen the Darwin type material. Although describing a species without seeing the type is inconsistent with current practices, it helps to remember that the perceived importance of type specimens has increased over the years and type specimens were not required for new species descriptions until 1935 (Turland et al. 2018, See Article 10.7), a result of the 1930 Cambridge Congress (Merrill 1930). Decaisne's description of C. chilensis could have been based upon the collections of Claudio Gay and Alcide d’Orbigny, two French naturalist contemporaries of Darwin who explored Chile and brought back their collections to PC (Fig. 5).   While Harvey (1849) made it clear from where specifically and generally C. chilensis specimens were collected, details regarding the material from Norfolk Island were vague. Montagne (1852) stated that he had personally not found C. chilensis, but clarified that it was Darwin who had found it on the coast of Chile, specifically in Valparaiso and in Puerto del   15 Hambre (Port Famine) in the Strait of Magellan, and off Norfolk Island, Australia (Montagne 1852, Appendix I, Fig. S5). Aside from Montagne’s report, C. chilensis was not reported on extensively until after Kützing’s (1858) publication nearly a decade after Harvey's publication.  Figure 5. Corallina sp. collected by Gay from Ancud, Chile in 1836 (A-C). See Appendix II, Table S1. The designation “paratype” is in error. Small pieces from this collection (D) were sent from the Paris museum herbarium for extraction by Jeffery Hughey.  BCA BAB CD  16 Nine years after C. chilensis Decaisne was published (Harvey, 1849), German phycologist Friedrich Traugott Kützing (1858 : 32) reduced C. chilensis to a variety, “Corallina officinalis chilensis,” (now Corallina officinalis var. chilensis (Decaisne) Kützing). Kützing's publication Tabulae Phycologicae; oder Abbildungen der Tange is a work of 8 volumes describing collections loaned to him by “foreign friends,” and its 8th volume (1858) emphasizes corallines. In this 8th volume, Kützing recognized eight varieties of C. officinalis in addition to C. officinalis var. chilensis from Chile, acknowledging the abundant intraspecific variation characteristic of corallines. The varieties were based on specimens from the North Sea, the Adriatic, and the Atlantic Ocean, and he attributed some of the variation to geographical location. Kützing considered C. officinalis chilensis (Fig. 6) to have been merely a Southern Hemisphere variety of C. officinalis. While some sources agreed with Kützing’s reduction in rank from species to variety, not all sources accepted the updated name. Thus both names, C. chilensis and C. officinalis var. chilensis, co-occur in the literature from 1858 onwards (Yendo 1902A, 1902B, Setchell & Gardener 1903, Skottsberg 1923, Dawson 1953, Papenfuss 1964, Ramírez & Santelices 1991, Hind & Saunders 2013A, Williamson et al. 2015).     17  Figure 6. Screenshot of Kützing’s 1858 description of C. officinalis [var.] chilensis, retrieved from AlgaeBase February 12, 2020. (A) Title page of publication “Illustrations of Seaweed.” (B) Sketch accompanying description. (a) Normal size (b) A small piece enlarged ~ 8x. (C) Latin description accompanying illustration.   There have been numerous reports of C. chilensis/C. officinalis var. chilensis since the late 1800’s, at first only in Chile. It was reported that C. officinalis var. chilensis was found in Magellanes province and Tierra del Fuego, Chile (Ardissone 1888), as well as in Bahia Orange, A BC  18 Chile (Hariot 1889, Appendix I, Fig. S6). Then, in 1901, Kichisaburo Yendo, a newly graduated Japanese phycologist, traveled to Canada and observed and then subsequently described the corallines growing near the Minnesota Seaside Station near Port Renfrew, BC, Canada (Yendo 1902A, Zasshi 1921). He reported that C. officinalis var. chilensis was present, although rare, and observed that it tended to “assume very diverse forms when found at the margins of the pools, or between tidal marks” (Yendo 1902A, Appendix I, Fig. S7). He remarked that the specimens fit better with Kützing’s illustration of C. officinalis var. chilensis than with Linnaeus’ C. officinalis and that it was also similar to specimens from Hakodate, Japan (Yendo 1902A). He thus believed that this southern hemisphere variety was present as far north as Vancouver Island, British Columbia, Canada and Japan. Unfortunately, Yendo fell ill and died at age 46 (Zasshi 1921). Many of the articulated coralline species that he described and illustrated (Yendo 1902A, 1902B) have not been found, making it impossible to verify his identifications. [Interestingly, Yendo (1902A) refers to the specimens from Canada as C. officinalis var. chilensis, and Yendo (1902B) refers to the specimens from Japan as C. chilensis.] Since Yendo’s 1902 reports, C. chilensis/C. officinalis var. chilensis has been extensively reported across both hemispheres, using morpho-anatomy to identify specimens. Setchell and Gardner (1903) reported that C. officinalis var. chilensis was rare further north in the East Pacific, but that it was commonly found on the coast of California (Appendix I, Fig. S8). Foslie (1907) reported finding young C. chilensis specimens in the Beagle channel “infested with Herposiphonia sullivana,” and in the Falklands, specifically Berkeley Sound, Port Louis (Appendix I, Fig. S9). Skottsberg (1923) likewise reported finding C. chilensis in tidepools in the Falklands, expressing that C. chilensis “quite possibly is only a form of C. officinalis, but further studies are necessary,” and he also reported “feather-like” branching (Appendix I, Fig. S10).   19 Skottsberg (1923) also included Japan and Peru in addition to Northwest America, Chile, and the Falklands with respect to C. chilensis’ distribution. Smith (1944) in his Marine Algae of the Monterey Peninsula reported that Corallina chilensis was “common everywhere” ranging from San Diego, California, north to Vancouver Island. He noted that it grew on rocks in the lower intertidal but was also found in tide pools higher in the intertidal, and that branches were in one plane and robust (Appendix I, Fig. S11).  Dawson (1953) provided a more detailed description of C. officinalis var. chilensis than any of the previous reports. He compared “common plants” from the Pacific Coast [assuming northern] with South American specimens and with C. officinalis specimens from all over the world noting that they all looked so similar, he did not think that Pacific American varieties were different species from the “classic” C. officinalis of the Northern Atlantic. The only difference he observed between North Atlantic Corallina officinalis and Pacific C. officinalis var. chilensis was the tendency of C. officinalis var. chilensis to be compound pinnate whereas North Atlantic C. officinalis was simple pinnate. Dawson (1953) also noted that C. officinalis var. chilensis grew “more abundantly” and “luxuriantly” in cooler waters over warmer waters, and that it commonly occurred all along the coast of Mexico, ranging from Isla Magdalena, Baja Mexico Sur, Mexico, north to British Columbia, Canada. Papenfuss (1964) noted that C. chilensis was distributed in the Falklands in his catalogue of Antarctic and sub-Antarctic benthic marine algae. Interestingly, while using the name “Corallina chilensis” instead of “Corallina officinalis var. chilensis,” Papenfuss also noted that Levring (1960) suspected that C. chilensis was not distinct from C. officinalis.  There are many reports of C. chilensis or C. officinalis var. chilensis having been collected from between southern Chile north through Lima, Peru between ~1900 and 1980   20 (Ramírez & Santelices 1991), as well as extensive reports from Mexico through British Columbia (Yendo 1902A, Setchell & Gardner 1903, Foslie 1907, Dawson 1953, Ramírez & Santelices 1991, Hind & Saunders 2013A).  Corallina officinalis var. chilensis has also been reported in South Africa (Silva et al. 1996). The reports of C. chilensis in the “arctic,” seem to refer to specimens collected in the Falklands and Southern Chile (Foslie 1907, Skottsberg 1923, Papenfuss 1964, Ramírez & Santelices 1991).  While there exist a great number of reports indicating that the species commonly occurred within the range of southern Chile through Peru, and from Baja California through British Columbia, Canada, it is important to note that all these historical reports were made exclusively based on morpho-anatomical comparisons, not DNA sequences.   In summary, (1) Corallina chilensis was originally published as a species that was later reduced to a variety, C. officinalis var. chilensis. (2) Kützing considered C. officinalis var. chilensis to be a southern variety of Northern Hemisphere C. officinalis based on morphology. (3) Yendo and others reported that this southern variety was also in the Northern Hemisphere (including British Columbia).   1.1.2 Study objectives  The following study objectives were motivated from the historical context surrounding the name C. officinalis var. chilensis. (1) Is C. chilensis a distinct species or should it be considered a variety, C. officinalis var. chilensis? (2) Is C. chilensis distributed in both hemispheres in the East Pacific?    21 1.2 Materials and methods   1.2.1 Sampling    I used a diversity of samples identified as Corallina. For the molecular-based portion of my study, I examined 77 specimens that were collected between 2007 and 2019 from Western Canada, the United States, Chile, Japan, Taiwan, and China, representing putative C. chilensis and other species. Details including location for the specimens may be found in Appendix II. Care was taken to include as many different species across various geographic locations as possible (Bergsten et al. 2012). Two of the samples were collected from the Biobio region of central Chile in 2019 and were field identified as C. officinalis var. chilensis, and an additional specimen collected in Chile (NCU 656905, Playa Cocholgue, Appendix II) was contributed by Paul Gabrielson. In addition to the 77 specimens from our contemporary collections, I included three specimens in this study that were collected in the 1830’s – 40’s. These were the Darwin specimen designated by Harvey as the type specimen of C. chilensis (2151, Valparaiso, Appendix II, Table S1), as well as the field identified C. chilensis collections of Darwin’s contemporaries Gay (Ancud, Chile, Appendix II, Table S1) and d’Orbigny (exact locality unknown, but thought to be Patagonia, see Appendix II, Table S1) which Decaisne may have used for his description of C. chilensis in the original name publication.   For the morphological-based portion of my study, I used 41 specimens collected between northern Oregon and northern British Columbia between 2007 and 2017 (Appendix II, Figure S10, Appendix III). Collections used for the morphological-based analysis overlapped with but were not identical to collections used for the molecular-based portion of this study.      22 1.2.2 DNA extraction, amplification, and sequence assembly    DNA was extracted following the red algal extraction protocol described in Hind et al. (2013A). Each ground sample was mixed with DNA extraction buffer, 10% Tween 20, and Proteinase K, and then treated using the Wizard® DNA extraction kit and eluted with fifty microliters of water or AE buffer.    Amplification targets were two chloroplast genes, psbA and rbcL and the mitochondrial COI (Cox1). These genes are commonly used for phylogenetic studies of coralline algae, and published DNA sequences are available for comparison (Hind et al. 2013A, Richards et al. 2017, Jeong et al. 2019, Twist et al. 2019). Including three genes can help to detect the issues that can complicate phylogenetic interpretation such as the retention of ancestral polymorphisms, hybridization, and incomplete lineage sorting that result in single gene trees inaccurately reflecting the speciation process (Leliaert et al. 2014). Amplification and one directional sequencing using either psbAF1 or psbAR2 was attempted for each DNA template to determine the success of extraction and for the purposes of confirming field identifications (Table 1). Templates of interest that were successfully sequenced in one direction using the psbA marker and were relatively free of contamination were then sequenced in forward and reverse directions for psbA, CO1, and rbcL genes (Table 1).     23 Table 1. Table of primer names, sequences, and sources for primers used in this analysis.   In preparation for PCR, DNA extract concentration was measured using a NanoDrop 8000 (Thermo Fisher Scientific, Wilmington, DE, United States), and diluted to a concentration of 40 to 80 ng/ml.   A master mix was prepared fresh as needed for each DNA template to be amplified plus enough excess for one positive control and one negative control. Each reaction contained 13.16 µl sterile water, 2.0 µl 10X Buffer (included with Taq), 1.6 µl 25 mM MgCl2, 1.6 µl 2.5 mM dNTPs, 0.28 µl 10 µM forward primer, 0.28 µl 10 µM reverse primer, and 0.09 µl 5U/µl Taq DNA polymerase (Invitrogen). For templates that failed to amplify, troubleshooting was attempted using puReTaqTM Ready-To-GoTM PCR beads following the manufacturer’s protocol.   PCR product was verified via gel electrophoresis on 1.0% agarose gels stained with 3.5 µl SYBR-Safe dye. (See Appendix IV, Table S6 for amplification thermal cycler conditions.) Successful amplification materials were stored in the -20oC freezer. Non-purified PCR product was sequenced by the McGill University and Genome Quebec Innovation Centre.   Chromatograms were imported into Geneious 7.1.9 (Biomatters Ltd., Auckland, New Zealand). Raw ends were trimmed to about 950 base pairs (bp) for psbA forward and reverse, 650-700 bp for CO1 forward and reverse, and 600-900 bp or 800-950 bp for rbcL. Forward and Gene Primer Direction Primer sequence SourcepsbAF1 Forward 5’ ATG ACT GCT ACT TTA GAA AGA CG 3’ Yoon et al. 2002psbAR2 Reverse 5’ TCA TGC ATW ACT TCC ATA CCT A 3’ Yoon et al. 2002GWSFn Forward 5’ TCA ACA AAY CAY AAA GAT ATY GG 3’ Le Gall & Saunders 2010GWSRx Reverse 5’ ACT TCT GGR TGI CCR AAR AAY CA 3’ Clarkston & Saunders 2012F57 Forward 5’ GTA ATT CCA TAT GCT AAA ATG GG 3’ Freshwater & Rueness 1994R1150K Reverse 5’ GCA TTT GAC CAC AAT GGA TAC 3’ Lindstrom et al. 2015F753 Forward 5’ GGA AGA TAT GTA TGA AAG AGC 3’ Freshwater & Rueness 1994rbcLrevNEW Reverse 5’ ACA TTT GCT GTT GGA GTY TC 3’ Kucera & Saunders 2012psbACO1rbcL  24 reverse sequences for each specimen were aligned, edited by eye and assembled into contigs, which were corrected manually to close gaps that had been inserted by single erroneous bases which tended to occur towards the ends of sequences, and to resolve ambiguous bases in highly conserved regions of the alignments. psbA sequences were 877 bp long, CO1 sequences were 680 bp long, and the majority of the rbcL sequences were 1334 bp long.    Jeffery Hughey (Department of Biology, Hartnell College) extracted and amplified DNA from the three 1800’s herbarium specimens (Appendix II, Table S1) following Hughey et al. 2001, as modified by Gabrielson et al. (2011), following recommendations by Hughey and Gabrielson 2012 and Saunders and McDevit 2012. A small portion of the rbcL gene was targeted using the F1152cor (Gabrielson et al. 2011) and R-rbcS (Freshwater and Rueness 1994) primers to produce 263 bp sequences. The Corallina chilensis type specimen (Darwin #2151), as well as specimens collected by Gay (Ancud specimen) and d’Orbigny, were sequenced in this way (Appendix II, Table S1).    Supplemental published sequences (See Appendix II, Table S1) were selected from the literature and retrieved from GenBank. These specific DNA sequences were chosen for the purpose of supplying outgroups (Appendix II, Table S2), confirming species identities, and maintaining consistency of species representation. Additional published sequences that were not included in the molecular analyses were used for confirming the contemporary range of Corallina chilensis (Appendix II, Table S3).     25 1.2.3 Sequence alignment & phylogenetic analysis  Three individual gene trees, a separate rbcL gene tree containing short DNA sequences from the 1800’s herbarium materials including the Darwin C. chilensis type, a majority rule tree, and a concatenated gene tree were generated during the phylogenetic analyses.   Individual gene trees & rbcL type tree   Edited Corallina sequences were aligned with published sequences including outgroups (Appendix II, Table S1). Sequences were placed in single-locus alignments using Geneious Prime® 2019.2.3, build 2019-09-24 (Biomatters Ltd., Auckland, New Zealand). The psbA alignment was composed of 91 sequences including outgroups, and was 851 aligned sites long. The CO1 alignment consisted of 63 sequences including outgroups, and was 660 aligned sites long. The rbcL alignment consisted of 47 sequences including outgroups, and was 1334 aligned sites long. The rbcL type alignment consisted of the same 47 sequences in the rbcL that were 1334 aligned sites long, along with 3 short 1800’s herbarium sequences of 263 aligned sites long.    Maximum likelihood trees were created in IQ-tree 1.6.12 for MacOSx (Nguyen et al. 2014) for each gene. Sequences were partitioned by codon position. Models of sequence evolution for each locus were estimated under Bayesian Information Criterion (BIC) utilizing ModelFinder (Kalyaanamoorthy et al. 2017) implemented in IQ tree (see table of evolution models implemented, Appendix IV, Table S7). Internal node robustness was assessed in IQ tree by 1,000 maximum likelihood bootstrap replicates and by approximate Likelihood Ratio Tests (aLRT) based Shimodaira-Hasegawa-like procedures (Anisimova 2006). MrBayes (Ronquist et al. 2011) was used to run Bayesian analyses on the three individual gene alignments. Since MrBayes has fewer sequence evolution models available than IQ-tree, ModelFinder in IQ-tree   26 was re-run on each partitioned dataset to determine the optimal sequence evolution models within the MrBayes available subset. (See Appendix IV, Table S7 for evolution models.) Two independent analyses were run on each partitioned dataset with four independent chains. Analyses ran for 4 million generations, sampled every 1,000 generations. The first 10% of the trees were discarded as burn-in, and trees from subsequent generations were saved because the log-likelihoods had plateaued after that point and estimated sample sizes of parameter values exceeded 200 when viewed in Tracer v1.7.1 (Rambaut et al. 2018). Trees were visualized using FigTree v1.4.4 (Rambaut 2018), and maximum likelihood bootstrap values and aLRT values were superimposed on the Bayesian tree topology.   Majority rule tree & concatenated trees   Individual gene trees revealed congruent species-level clades but unresolved, confusing, and incongruent relationships among species. I performed two additional phylogenetic analyses to evaluate congruence and incongruence among single gene trees (Maddison 1997, Mossel & Vigoda 2005, Liu & Pearl 2007). The first involved comparing clades appearing in majority rule bootstrap consensus trees from each locus. For each locus, 1000 bootstrap trees were generated in RAxMLGUI 1.5 beta (Silvestro & Michalak, 2012), using all PTM and published sequences listed in Appendix II, Tables S1 & S2, except for PTM 826, the 1800’s herbarium materials, or as otherwise noted in Table S1. For each locus, a 50% majority rule consensus tree was created from the 1000 bootstrap trees using PAUP Version 4.0a, build 167 (Sunderland, Massachusetts, USA; Swofford 2002). A final majority rule consensus tree was then created in PAUP from the three majority rule individual gene consensus trees.    27   A second phylogenetic analysis explored the incongruence among sequences from different loci from Corallina officinalis within a tree from a concatenated alignment. The alignment, created in Geneious, included the concatenated gene sequences available from each voucher listed in Appendix II, Table S2. For Corallina officinalis, each psbA, rbcL and CO1 sequence was added as a separate operational taxonomic unit, without concatenating different genes from the same voucher. To confirm that the manual alignment using Geneious was not responsible for incongruences among loci, all concatenated sequences were realigned in MAFFT version 7 (Katoh 2013). The alignment was partitioned by codon and GTR gamma + I substitution model was used in creating the concatenated tree.    The concatenated gene tree was the most likely from 200 replicated searches in RAxML-HPC2 on XSEDE through the CIPRES Science Gateway V 3.3. (Miller et al. 2010). The alignment was analyzed in RAxMLGUI to produce 1000 bootstrap trees and bootstrap percentages which were then overlaid on the most likely tree.   1.2.4 Other analyses supporting species delimitation    Automatic Barcode Gap Discovery (ABGD) for delimitation of candidate species (Puillandre et al. 2011) was applied to the psbA, CO1, and rbcL alignments. The ABGD barcoding analysis may only be performed on single gene alignments, so there was no analysis of the concatenated alignment. ABGD was run with P-min set to 0.001 and P-max set to 0.1, steps set to 10, Nb bins set to 20, X relative gap width equal to 1.5, and the Jukes-Candor (J669) option selected. Output partitions were chosen based on how well they fit to currently recognized Corallina species delimitations, prioritizing partitions that grouped C. vancouveriensis as a distinct species from C. officinalis (Hind & Saunders 2013A).    28 Uncorrected pairwise percent differences between sequences were generated in Geneious Prime® 2019.2.3. For the purpose of maintaining consistency between these results and other studies, genetic distances throughout the methods, discussion, and future directions portion of this thesis were compared with distances in Hind et al. 2018 and references therein (Broom et al. 2008, Hind and Saunders 2013A, Nelson et al. 2015, Hind et al. 2016, Hind et al. 2018). These distances were 0.7-1.3 % difference between species in psbA, 4.5-5.8% difference between species in CO1, and 1.6-1.9% difference between species in rbcL (Hind et al. 2018). These percent differences differed from earlier papers with lower thresholds that will also appear later in the discussion. Three DNA sequences corresponding with specimens that were interesting because of their geographical origin and that were thought to have been Corallina chilensis were contributed too late to be included in my phylogenetic analysis. Two of the specimens were collected in 2019 (Biobio, Chile) and identified in the field as C. officinalis var. chilensis, and one was a recent collection from Chile contributed by Paul Gabrielson. I tested their identity based on previous sequencing results and by comparison to sequences in GenBank (Appendix II, Table S3). I considered that specimens might be conspecific if their sequences shared at least 75% coverage and were at least 98% similar for psbA, 98.5% similar for CO1, and 99% similar for rbcL.   1.2.5 Morphometric analysis     Morphology of Corallina sp. 1 frondescens collections from British Columbia was analyzed because BLAST searches with preliminary DNA sequence data suggested that they might be conspecific with C. chilensis (Appendix III). A morphological analysis of Northeast Pacific C. vancouveriensis also appears in this chapter because C. vancouveriensis grows   29 abundantly along-side C. sp. 1 frondescens across its entire Northeast Pacific range. Appendix III lists the specimens used for this analysis. The majority of C. sp. 1 frondescens specimens had multiple fronds per specimen, so one individual frond was selected arbitrarily from each specimen for measurement. Measurements included in the morphometric analysis are summarized in Fig. 7. Height and maximum width of each frond were measured, as was length of the crown and length of the stem (Fig. 7A-C). The crown was defined as the branching upper portion of the main axis with branches consisting of more than one intergeniculum per branch. (Basal branchlets, only one intergeniculum long were discounted.) The stem was defined as the region of the main axis starting from the most basal unbranched intergeniculum to the branch inflection point (Fig. 7C). On the same frond, the length of a secondary pinnate branch off the main axis was arbitrarily selected for measurement (Fig. 7D). Intergenicular dimensions were measured on haphazardly chosen mid-intergenicula from randomly selected main axes and secondary branches for each collection (Fig. 7D-F). Arbitrarily selected basal intergenicula from the main axes were measured (Fig. 7G) as well as conceptacle branches (including the subtending intergenicula) when present in the sample (Fig. 7H). Because it was often unclear if smaller fronds were branches from larger fronds within the same sample or if they were independent individuals, the frond length and width at the widest point was also measured from the tallest frond of each specimen to ensure that maximum height and width of frond data would not be deflated.    Photographs were taken of six representative C. sp. 1 frondescens (Appendix II, Tables S1 & S3) specimens from British Columbia. Two were collected from Botany Beach, Vancouver Island, and the other four are herbarium specimens collected from the Hakai conservancy on Calvert Island.   30  Figure 7. Summary of measurements taken for morphometric analysis. Generic Corallina illustrations modeled after C. sp. 1 frondescens not drawn to scale. (A) Tallest frond length and width. (B) Random frond length and width. (C) Crown length and stem length. (D) Secondary branch length. (E) Main axis mid intergenicular dimensions. (F) Secondary branch mid intergenicular dimensions. (G) Basal intergenicular dimensions on main axis. (H) Conceptacle branch dimensions. Length includes subtending intergeniculum and bulbous head.               minmaxminmaxA BC D EF G H  31 1.3 Results     Table 2 shows the preliminary Automatic Barcode Gap Discovery (ABGD) species delimitation and the following figures (Figs. 8-22) include all phylogenetic trees described in the methods (see Methods 1.2.3). Figures 8-9 show an rbcL gene tree that includes sequences from mid-1800's herbarium specimens. The next three phylogenies (Figs. 10-15) are individual psbA (Figs. 10-11), CO1 (Figs. 12-13), and rbcL (Figs. 14-15) trees that do not contain sequences from the 1800’s herbarium material. The individual gene trees are followed by the majority rule tree (Figs. 16-19) illustrating the disagreement across all three individual gene trees. The final tree is the concatenated tree (Figs. 20-22). The majority rule tree (and the concatenated tree likewise do not contain any sequences from 1800’s herbarium material.               32   Table 2. Side by side results of three ABGD barcoding analyses performed on psbA, CO1, and rbcL single gene alignments. Boxes indicate species as determined by each analysis. The psbA analysis identified 13 species, the CO1 analysis 23 species, and the rbcL analysis 16 species.  psbA analysis CO1 analysis rbcL analysisC. officinalis C. officinalis C. officinalisC. sp. 1 california C. sp. 1 california C. sp. 1 californiaC. chilensis C. chilensis C. chilensisC. sp. 2 frondescens C. sp. 2 frondescens C. sp. 5 frondescensC. sp. 5 frondscens C. sp. 5 fondescens C. sp. 2 frondescensC. sp. 3 frondescens C. sp. 3 frondescens C. sp. 3 frondescensC. sp. 3 frondescens-like C. sp. 3 frondescens, PTM1400 C. sp. 3 frondescens-likeC. sp. 2 vancouveriensis C. sp. 3 frondescens-like C. sp. 1 gwsC. maxima C. sp. 2 vancouveriensis C. sp. 1 gws-likeC. sp. 1 gws C. maxima C. ferreyrae-likeC. sp. 1 gws-like C. sp. 1 gws C. vancouveriensisC. ferreyrae-like C. sp. 1 gws-like C. sp. 1 chileC. declinata C. ferreyrae-like C. sp. 4 frondescensC. aberrans C. declinata C. ferreyrae  (Bustamante)C. vancouveriensis C. aberrans C. ferreyrae (PTM826 only)C. sp. 1 chile C.  vancouveriensis C. sp. 2 chileC. sp. 4 frondescens C. sp. 1 chile C. sp. 2 chileC. ferreyrae (Bustamante) C. sp. 4 frondescens C. pinnatifolia C. ferreyrae (PTM) C. sp. 4 frondescens C. crassissimaC. sp. 2 chile C. ferreyrae (PTM 826 only) C. aberransC. crassissima C. ferreyrae (Bustamante) C. melobesioidesC. ferreyrae (PTM 819 only)C. sp. 2 chileC. caespitosa holotypeC. crassissimaC. sp. 5 korea  33  Figure 8. rbcL type tree. Entire phylogenetic rbcL tree including Corallina chilensis type specimen collected by Darwin. Asterisks designate 263 bp sequences of herbarium material from the 1800’s, from the type and from specimens from d’Orbigny and Gay C. chilensis; and type specimens included in this tree. Top two branch support values are aLRT/maximum likelihood bootstrap percentages. The bottom value is the Bayesian posterior probability. The scale bar refers to substitutions per site and the blue box indicates the portion of the tree that is expanded in Fig. 9.     rbcL type tree0.05chilensisDarwinlikecaespitJapan_PTM1408like1gwsJapan_PTM1409CoffAlaskaNCU588445_KJ591672h14sp1caliCanada_PTM1188OUTEllisBM000806006KP834400Williams15chilensisGayCoff_epitypeBM001062598JX315329Brod13sp4Chile_PTM844sp1chileChile_PTM1337sp1chileChile_PTM899OUTcrusticatUBCa89963KU983253h16sp5Canada_PTM420chilensisdOrbignysp2chileChile_PTM870OUTCallicheiloHQ322294gab11CvanCanada_PTM767crassJapan_PTM_1490sp1Canada_PTM332declinaberransJapan_PTM1445sp1chileChile_PTM910sp2Canada_PTM1178caespitChile_PTM826sp1caliCanada_PTM363sp1gwsJapan_PTM1457likecaespitJapan_PTM1416sp2chileCalifornia_PTM1254OUTlithoglas_KC134336h18sp4OregonPTM1235ferreyraeMK408748Cpinn_topotypeCaliforniaA88590HQ322333ptm12sp1caliCalifornia_PTM1247likecaespit_Japan_PTM1440CoffBM001004107_JN701476h14likesp3Japan_PTM1419sp3Japan_PTM1405sp1caliCanada_PTM515sp1chileChile_PTM1325like1gwsJapan_PTM1401OUTfrondUBCa90727KT782137h15sp3Japan_PTM1442sp1OregonPTM1244CoffCanadaNCU590595KJ591674h14like1gwsJapan_PTM1402sp1chileChile_PTM863melobesioidesJapan_UBC_A62034_JN701477ptm12OUTChibodJQ677000hs13othersp3Japan_PTM1400crassJapan_PTM1447likesp3Japan_PTM1439Litho amnion glaciale Newfoundland & Labrador, Canada KC134336Ellisolandia elongata Llanes, Asturias, Spain  KP834400Calliarthron cheilosporioides California, United States HQ322294Bossiella frondifera  Bamfield, British Columbia, Canada KT782137Chiharaea bodegensis  Canadian Northeast Pacific JQ677000Crusticorallina muricata  Botany Bay, British Columbia, Canada KU983253C. sp. 3 frondescens-like  Muroran, Japan  PTM1419C. sp. 3 fro descens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  Marine Station, Oshoro, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442*C. officinalis epitype Devon, England JX315329. officinalis Somerset, England JN701476C. officinalis  Alaska, United States KJ591672C. officinalis Foster Island, British Columbia, Canada KJ591674C. vancouveriensis Hakai, British Columbia, Canada  PTM767C. sp. 1 california  California, United States PTM1247C. sp. 1 california  Hakai, British Columbia, Canada PTM515C. sp. 1 california Hakai, British Columbia, Canada PTM363C. sp. 1 california  Hakai, British Columbia, Canada PTM1188C. sp. 5 frondescens  Hakai, British Columbia, Canada PTM420C. sp. 4 frondescens  Valparaiso, Chile PTM844C. sp. 4 frondescens  Oregon, United States PTM1235C. sp. 2 frondescens  Hakai, British Columbia, Canada PTM1178C. sp. 1 frondescens  Hakai, British Columbia, Canada PTM332*C. chilensis Darwin type sequence Valparaiso, Chile*C. chilensis d’Orbigny historical material sequence Chile C. sp. 1 frondescens Oregon, United States PTM1244C. aberrans Katsuura, Japan  PTM1445C. crassissima  Kominato Kamogawa Chiba-ken, Japan PTM1490C. crassissima  Marine Institute, Katsuura, Japan  PTM1447C. sp. 1 gws  Katsuura, Japan PTM1457C. sp. 1 gws-like  arine Station, Oshoro Bay, Japan PTM1402C. sp. 1 gws-like arine Station, Oshoro Bay, Japan PTM1401C. sp. 1 gws-like  arine Station, Oshoro Bay, Japan PTM1409C. ferreyrae  Quintay, Chile PTM826*C. fe r yrae isotype Pucusana, Peru MK408748C. ferreyrae-like  Cape Tachimachi, Hakodate, Japan PTM1440C. ferreyrae-like  Marine Station, Oshoro Bay, Japan PTM1408C. ferreyrae-like  Muroran, Japan PTM1416*C. chilensis Gay historical material sequence ChileC. sp. 2 c ile  Curinaco, Chile  PTM870C. melobesioides  Awa-Kominato, Chiba-ken, Japan JN701477C. pinnatifolia  California, United States HQ322333C. sp. 2 chile  C lifornia, United States PTM1254C. sp. 1 c il   Bonifacio, Chile PTM899C. sp. 1 c ile  Curinaco, Chile PTM863C. sp. 1 c il   Mar Brava, Chile PTM910C. sp. 1 c   Cucao, Chile PTM1325C. sp. 1 c il   ucatrihue, Chile PTM133796.9/98190.5/81194.3/89196.6/77195.5/99186.8/92196.7/99188.5/63187.2/56197.1/62-93.1/80.98100/100173/66.8476.2/30.54100/100192.1/100199.5/99196.4/100187.9/94199.5/100181.5/29.7799/74.9429.3/63.8887.1/84.98100/1001Figure 8rbcL tree with C. chilensis type sequencesGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesianExpanded in Fig. 9  34  Figure 9. Expanded portion of the rbcL type tree from Fig. 8.   rbcL type tree0.05chilensisDarwinlikecaespitJapan_PTM1408like1gwsJapan_PTM1409CoffAlaskaNCU588445_KJ591672h14sp1caliCanada_PTM1188OUTEllisBM000806006KP834400Williams15chilensisGayCoff_epitypeBM001062598JX315329Brod13sp4Chile_PTM844sp1chileChile_PTM1337sp1chileChile_PTM899OUTcrusticatUBCa89963KU983253h16sp5Canada_PTM420chilensisdOrbignysp2chileChile_PTM870OUTCallicheiloHQ322294gab11CvanCanada_PTM767crassJapan_PTM_1490sp1Canada_PTM332declinaberransJapan_PTM1445sp1chileChile_PTM910sp2Canada_PTM1178caespitChile_PTM826sp1caliCanada_PTM363sp1gwsJapan_PTM1457likecaespitJapan_PTM1416sp2chileCalifornia_PTM1254OUTlithoglas_KC134336h18sp4OregonPTM1235ferreyraeMK408748Cpinn_topotypeCaliforniaA88590HQ322333ptm12sp1caliCalifornia_PTM1247likecaespit_Japan_PTM1440CoffBM001004107_JN701476h14likesp3Japan_PTM1419sp3Japan_PTM1405sp1caliCanada_PTM515sp1chileChile_PTM1325like1gwsJapan_PTM1401OUTfrondUBCa90727KT782137h15sp3Japan_PTM1442sp1OregonPTM1244CoffCanadaNCU590595KJ591674h14like1gwsJapan_PTM1402sp1chileChile_PTM863melobesioidesJapan_UBC_A62034_JN701477ptm12OUTChibodJQ677000hs13othersp3Japan_PTM1400crassJapan_PTM1447likesp3Japan_PTM1439Litho amnion glaciale Newfoundland & Labrador, Canada KC134336Ellisolandia elongata Llanes, Asturias, Spain  KP834400Calliarthron cheilosporioides California, United States HQ322294Bossiella frondifera  Bamfield, British Columbia, Canada KT782137Chiharaea bodegensis  Canadian Northeast Pacific JQ677000Crusticorallina muricata  Botany Bay, British Columbia, Canada KU983253C. sp. 3 frondescens-like  Muroran, Japan  PTM1419C. sp. 3 fro descens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  Marine Station, Oshoro, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442*C. officinalis epitype Devon, England JX315329. officinalis Somerset, England JN701476C. officinalis  Alaska, United States KJ591672C. officinalis Foster Island, British Columbia, Canada KJ591674C. vancouveriensis Hakai, British Columbia, Canada  PTM767C. sp. 1 california  California, United States PTM1247C. sp. 1 california  Hakai, British Columbia, Canada PTM515C. sp. 1 california Hakai, British Columbia, Canada PTM363C. sp. 1 california  Hakai, British Columbia, Canada PTM1188C. sp. 5 frondescens  Hakai, British Columbia, Canada PTM420C. sp. 4 frondescens  Valparaiso, Chile PTM844C. sp. 4 frondescens  Oregon, United States PTM1235C. sp. 2 frondescens  Hakai, British Columbia, Canada PTM1178C. sp. 1 frondescens  Hakai, British Columbia, Canada PTM332*C. chilensis Darwin type sequence Valparaiso, Chile*C. chilensis d’Orbigny historical material sequence Chile C. sp. 1 frondescens Oregon, United States PTM1244C. aberrans Katsuura, Japan  PTM1445C. crassissima  Kominato Kamogawa Chiba-ken, Japan PTM1490C. crassissima  Marine Institute, Katsuura, Japan  PTM1447C. sp. 1 gws  Katsuura, Japan PTM1457C. sp. 1 gws-like  arine Station, Oshoro Bay, Japan PTM1402C. sp. 1 gws-like arine Station, Oshoro Bay, Japan PTM1401C. sp. 1 gws-like  arine Station, Oshoro Bay, Japan PTM1409C. ferreyrae  Quintay, Chile PTM826*C. fe r yrae isotype Pucusana, Peru MK408748C. ferreyrae-like  Cape Tachimachi, Hakodate, Japan PTM1440C. ferreyrae-like  Marine Station, Oshoro Bay, Japan PTM1408C. ferreyrae-like  Muroran, Japan PTM1416*C. chilensis Gay historical material sequence ChileC. sp. 2 c ile  Curinaco, Chile  PTM870C. melobesioides  Awa-Kominato, Chiba-ken, Japan JN701477C. pinnatifolia  California, United States HQ322333C. sp. 2 chile  C lifornia, United States PTM1254C. sp. 1 c il   Bonifacio, Chile PTM899C. sp. 1 c ile  Curinaco, Chile PTM863C. sp. 1 c il   Mar Brava, Chile PTM910C. sp. 1 c   Cucao, Chile PTM1325C. sp. 1 c il   ucatrihue, Chile PTM133796.9/98190.5/81194.3/89196.6/77195.5/99186.8/92196.7/99188.5/63187.2/56197.1/62-93.1/80.98100/100173/66.8476.2/30.54100/100192.1/100199.5/99196.4/100187.9/94199.5/100181.5/29.7799/74.9429.3/63.8887.1/84.98100/1001Figure 9rbcL tree with C. chilensis type sequencesGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian  35  Figure 10. Entire phylogenetic tree of 91 psbA sequences from Corallina specimens and six outgroups. The top two branch support values are aLRT/maximum likelihood bootstrap percentages. The bottom number is the Bayesian posterior probability. The scale bar indicates substitutions per site, and the blue box is the portion of the tree expanded in Fig. 16. Asterisks denote type sequences.  0.041GWS_Japan_GWS013769JQ422217HS2013caespit_Chile_PTM827OutCalcheilospr_JQ422199HS2013sp1_Canada_PTM1821chile_Chile_PTM8622chile_MChile_PTM905ferreyrae_Peru_MK408748sp3_Japan_PTM1405likecaespit__Japan_PTM14161chile_Chile_PTM8892chile_USA_PTM1265Cvan_Canada_PTM767likecaespit_Japan_PTM1440caespit_Chile_PTM819sp4_Chile_PTM8222chile_Taiwan_PTM1519Cvan_Canada_PTM760CoffCanada_NCU590595KJ637652Hind20142chile_USA_PTM1254cali_USA_GWS021316JQ422238HS20132chile_USA_PTM1262sp2van_Canada_GWS009913JQ422229HS2013likecaespitJapan_PTM1417OUTlitho_KP224290Hind2018crassissima_Japan_GWS013776_JQ422203OutChibodeg_JQ677009_HS2013otherdeclinata__Japan_GWS013767_JQ422204_HS20131chile_Chile_PTM899caespit_Chile_PTM830caespit_Chile_PTM8472chile_Chile_PTM867sp4_Canada_GWS010351JQ422222HS2013caespit_Chile_PTM832sp5_Canada_GWS012660JQ422227HS20131chile_Chile_PTM868crassissima_Japan_PTM14901chile_Chile_PTM869sp1_Canada_PTM738likesp3_Japan_PTM1419sp1_Canada_PTM7891chile_Chile_PTM910sp3_Canada_GWS006466JQ422221HS2013sp4_Chile_PTM844CoffAlaska_NCU588445KJ637651Hind20141chile_Chile_PTM891caespit_Chile_PTM8211chile_Chile_PTM863sp4_Chile_PTM846caespit_Chile_PTM833cali_USA_PTM1247CoffCanada_GWS006989JQ422209HS2013sp5_GWS006561JQ422226HS2013aberrans_Japan_PTM1445sp3_Japan_PTM1442sp4_Chile_PTM8811chile_Chile_PTM926OutCrustimur_UBCa89963KU983300Hind20162chile_Chile_PTM880sp1_Oregon_PTM1244sp1_Canada_PTM740likeGWS_Japan_PTM1401cali_Canada_PTM5152chile_Chile_PTM870sp2_Canada_PTM489OutEllisGWS001818_JQ422231aberrans_Japan_GWS013777_JQ422201_HS2013sp1_Canada_PTM742Cvan_Canada_GWS010831JQ422228HS2013CoffEngland_BM001004107JQ917413Hind2014Merwe2015cali_Canada_PTM1188declinata_Japan_PTM1488sp1_Canada_PTM743sp1_Canada_PTM7881chile_Chile_PTM879maximasyntype__Japan_GWS013782_JQ4222072chile_Chile_PTM8952chile_USA_PTM1266OUT_Bfrondif_UBCa90727KT782243Hind2015sp3_Japan_PTM14001GWS_Japan_PTM1457sp4_Chile_PTM842sp4_Oregon_USA_PTM1235cali_Canada_PTM3631chile_Chile_PTM898sp2_Canada_PTM1178sp5_Canada_PTM4201chile_Chile_PTM876caespit_Chile_PTM826sp1_Canada_PTM3322chile_Chile_PTM873likesp3_Japan_PTM1439Lithothamnion glaciale  Newfoundland & Labrador, Canada KP224290 Ellisolandia elongata  Leitrim, Ireland JQ422231 Calli rt r n cheilosporioides  British Columbia, Canada JQ422199Bossiella frondifera  Bamfield, British Columbia, Canada KT782243Chiharaea bodegensis  Northeast Pacific Canada? JQ677009Crusticorallina muricata  Botany Bay, British Columbia, Canada KU983300C. aberrans  Chiba-ken, Japan  JQ42 201C. aberrans Katsuura, Japan  PTM1445C. declinata  Chiba-ken, Japan  JQ422204C. declinata  Chiba-ken, Japan  PTM1488C. cra sissim   Chiba-ken, apan  JQ422203C. cra sissim   Chiba-ken, Japan  PTM1490C. sp. 3 frondescens-like  Muroran, Japan  PTM1419C. s . 3 fro descens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  British Columbia, Canada  JQ422221C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM489C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. maxima Chiba-ken, Japan  JQ42 0C. sp. 1 gws  Chiba-ken, Japan  JQ422217C. sp. 1 gws  Katsuura, Japan  PTM1457C. sp. 1 gws-like  Marine Station, Oshoro Bay, Japan  PTM1401C. sp. 2 vancouveriensis  British Columbia, Canada  JQ422229. vancouveriensis  British Columbia, Canada  JQ422228C. vancouveriensis  Hakai, British Columbia, Canada  PTM760. vancouveriensis  Hakai, British Columbia, Canada  PTM767C. sp. 4 frondescens  British Columbia, Canada  JQ422222C. sp. 4 frondescens  Quintay, Chile  PTM822C. sp. 4 frondescens  Valparaiso, Chile  PTM842C. sp. 4 frondescens  Valparaiso, Chile  PTM844C. sp. 4 frondescens  Valparaiso, Chile  PTM846C. sp. 4 frondescens  Curinaco, Chile  PTM881C. sp. 4 frondescens  Oregon, United States  PTM1235C. sp. 5 frondescens  British Columbia, Canada  JQ422227C. sp. 5 frondescens  Hakai, British Columbia, Canada  PTM420C. sp. 5 frondescens  British Columbia, Canada  JQ422226C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM363C. sp. 1 california  Hakai, British Columbia, Canada PTM 515 PTM515C. sp.  c lifornia  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california  California, United States  JQ422238C. sp. 1 california  California, United States  PTM1247C. chilensis  Hakai, British Columbia, Canada  PTM332C. chilensis  Bamfield, British Columbia, Canada  PTM740C. chilensis  Bamfield, British Columbia, Canada  PTM743C. chilensis  Bamfield, British Columbia, Canada PTM738C. chilensis  Bamfield, British Columbia, Canada  PTM742C. chilensis Hakai, British Columbia, Canada  PTM789C. chilensis  Port Renfrew, British Columbia, Canada  PTM182C. chilensis  Hakai, British Columbia, Canada PTM788C. chilensis  Oregon, United States  PTM1244. officinalis  Alaska, United States  KJ637651C. officinalis  Newfoundland & Labrador, Canada  JQ422209. officin lis  Foster Island, British Columbia, Canada  KJ637652. officinalis  Somerset, England  JQ917413C. sp. 1 chile  Curinaco, Chile  PTM862C. sp. 1 chile  Curinaco, Chile  PTM863C. sp. 1 chile  Curinaco, Chile  PTM868C. sp. 1 chile  Curinaco, Chile  PTM869C. sp. 1 chile  Curinaco, Chile  PTM876C. sp. 1 chile  Curinaco, Chile  PTM879C. sp. 1 chile  Bonifacio, Chile  PTM889C. sp. 1 chile  Bonifacio, Chile  PTM898C. sp. 1 chile  ar Brava, Chile  PTM910C. sp. 1 chile  Cucao, Chile  PTM926C. sp. 1 chile  Bonifacio, Chile  PTM891C. sp. 1 chile  Bonifacio, Chile  PTM899C. ferreyrae-like Cape Tachimachi, Hakodate, Japan  PTM1440C. ferreyrae-like  Muroran, Japan  PTM1416C. ferreyr e-like  uroran, Japan  PTM1417C. ferreyrae  Quintay, Chile  PTM819C. ferreyrae  Quintay, Chile  PTM821C. ferreyrae  Quintay, Chile  PTM826C. ferreyrae  Quintay, Chile  PTM827C. ferreyrae  Quintay, Chile  PTM830C. ferreyrae  Quintay, Chile  PTM832C. ferreyrae  Quintay, Chile  PTM833C. ferreyrae  Valparaiso, Chile  PTM847*C. ferreyrae  isotype Pucusana, Peru  MK408748C. sp. 2 chile  Curinaco, Chile  PTM867C. sp. 2 chile  Curinaco, Chile  PTM870C. sp. 2 chile  Curinaco, Chile  PTM873C. sp. 2 chile  Curinaco, Chile  PTM880C. sp. 2 chile  Bonifacio, Chile  PTM895C. sp. 2 c ile  Mar Brava, Chile  PTM905C. sp. 2 chile  Keelung, Taiwan  PTM1519C. sp. 2 chile  California, United States  PTM1254C. sp. 2 chile  California, United States  PTM1262C. sp. 2 chile  California, United States  PTM1265C. sp. 2 chile  California, United States  PTM1266100/99.9193.9/98190/981-/-.96-/-.8681.8/84.8689.4/55.97 97.3/98196.9/88185.8/99175.5/28.7596.1/97184.9/57.8694.5/73190/92.9777.3/5.6492.3/95186.1/22.9595.1/100187.5/43.9974.5/39.7695.9/84144.2/58.93psbA treeGenus CorallinaaLRT & 1000 ML percentagesBayesian posterior probabilitiesaLRT/MLBayesianExpanded in Fig. 11  36  Figure 11. This has been expanded from the psbA tree in Fig. 10   0.041GWS_Japan_GWS013769JQ422217HS2013caespit_Chile_PTM827OutCalcheilospr_JQ422199HS2013sp1_Canada_PTM1821chile_Chile_PTM8622chile_MChile_PTM905ferreyrae_Peru_MK408748sp3_Japan_PTM1405likecaespit__Japan_PTM14161chile_Chile_PTM8892chile_USA_PTM1265Cvan_Canada_PTM767likecaespit_Japan_PTM1440caespit_Chile_PTM819sp4_Chile_PTM8222chile_Taiwan_PTM1519Cvan_Canada_PTM760CoffCanada_NCU590595KJ637652Hind20142chile_USA_PTM1254cali_USA_GWS021316JQ422238HS20132chile_USA_PTM1262sp2van_Canada_GWS009913JQ422229HS2013likecaespitJapan_PTM1417OUTlitho_KP224290Hind2018crassissima_Japan_GWS013776_JQ422203OutChibodeg_JQ677009_HS2013otherdeclinata__Japan_GWS013767_JQ422204_HS20131chile_Chile_PTM899caespit_Chile_PTM830caespit_Chile_PTM8472chile_Chile_PTM867sp4_Canada_GWS010351JQ422222HS2013caespit_Chile_PTM832sp5_Canada_GWS012660JQ422227HS20131chile_Chile_PTM868crassissima_Japan_PTM14901chile_Chile_PTM869sp1_Canada_PTM738likesp3_Japan_PTM1419sp1_Canada_PTM7891chile_Chile_PTM910sp3_Canada_GWS006466JQ422221HS2013sp4_Chile_PTM844CoffAlaska_NCU588445KJ637651Hind20141chile_Chile_PTM891caespit_Chile_PTM8211chile_Chile_PTM863sp4_Chile_PTM846caespit_Chile_PTM833cali_USA_PTM1247CoffCanada_GWS006989JQ422209HS2013sp5_GWS006561JQ422226HS2013aberrans_Japan_PTM1445sp3_Japan_PTM1442sp4_Chile_PTM8811chile_Chile_PTM926OutCrustimur_UBCa89963KU983300Hind20162chile_Chile_PTM880sp1_Oregon_PTM1244sp1_Canada_PTM740likeGWS_Japan_PTM1401cali_Canada_PTM5152chile_Chile_PTM870sp2_Canada_PTM489OutEllisGWS001818_JQ422231aberrans_Japan_GWS013777_JQ422201_HS2013sp1_Canada_PTM742Cvan_Canada_GWS010831JQ422228HS2013CoffEngland_BM001004107JQ917413Hind2014Merwe2015cali_Canada_PTM1188declinata_Japan_PTM1488sp1_Canada_PTM743sp1_Canada_PTM7881chile_Chile_PTM879maximasyntype__Japan_GWS013782_JQ4222072chile_Chile_PTM8952chile_USA_PTM1266OUT_Bfrondif_UBCa90727KT782243Hind2015sp3_Japan_PTM14001GWS_Japan_PTM1457sp4_Chile_PTM842sp4_Oregon_USA_PTM1235cali_Canada_PTM3631chile_Chile_PTM898sp2_Canada_PTM1178sp5_Canada_PTM4201chile_Chile_PTM876caespit_Chile_PTM826sp1_Canada_PTM3322chile_Chile_PTM873likesp3_Japan_PTM1439Lithothamnion glaciale  Newfoundland & Labrador, Canada KP224290 Ellisolandia elongata  Leitrim, Ireland JQ422231 Calli rt r n cheilosporioides  British Columbia, Canada JQ422199Bossiella frondifera  Bamfield, British Columbia, Canada KT782243Chiharaea bodegensis  Northeast Pacific Canada? JQ677009Crusticorallina muricata  Botany Bay, British Columbia, Canada KU983300C. aberrans  Chiba-ken, Japan  JQ42 201C. aberrans  Katsuura, Japan  PTM1445C. declinata  Chiba-ken, Japan  JQ422204C. declinata  Chiba-ken, Japan  PTM1488C. cra sissim   Chiba-ken, apan  JQ422203C. cra sissim   Chiba-ken, Japan  PTM1490C. sp. 3 frondescens-like  Muroran, Japan  PTM1419C. s . 3 fro descens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  British Columbia, Canada  JQ422221C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM489C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. maxima Chiba-ken, Japan  JQ42 0C. sp. 1 gws  Chiba-ken, Japan  JQ422217C. sp. 1 gws  Katsuura, Japan  PTM1457C. sp. 1 gws-like  Marine Station, Oshoro Bay, Japan  PTM1401C. sp. 2 vancouveriensis  British Columbia, Canada  JQ422229. vancouveriensis  British Columbia, Canada  JQ422228C. vancouveriensis  Hakai, British Columbia, Canada  PTM760. vancouveriensis  Hakai, British Columbia, Canada  PTM767C. sp. 4 frondescens  British Columbia, Canada  JQ422222C. sp. 4 frondescens  Quintay, Chile  PTM822C. sp. 4 frondescens  Valparaiso, Chile  PTM842C. sp. 4 frondescens  Valparaiso, Chile  PTM844C. sp. 4 frondescens  Valparaiso, Chile  PTM846C. sp. 4 frondescens  Curinaco, Chile  PTM881C. sp. 4 frondescens  Oregon, United States  PTM1235C. sp. 5 frondescens  British Columbia, Canada  JQ422227C. sp. 5 frondescens  Hakai, British Columbia, Canada  PTM420C. sp. 5 frondescens  British Columbia, Canada  JQ422226C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM363C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM515C. sp.  c lifornia  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california  California, United States  JQ422238C. sp. 1 california  California, United States  PTM1247C. chilensis  Hakai, British Columbia, Canada  PTM332C. chilensis  Bamfield, British Columbia, Canada  PTM740C. chilensis  Bamfield, British Columbia, Canada  PTM743C. chilensis  Bamfield, British Columbia, Canada PTM738C. chilensis  Bamfield, British Columbia, Canada  PTM742C. chilensis Hakai, British Columbia, Canada  PTM789C. chilensis  Port Renfrew, British Columbia, Canada  PTM182C. chilensis  Hakai, British Columbia, Canada PTM788C. chilensis  Oregon, United States  PTM1244. officinalis  Alaska, United States  KJ637651C. officinalis  Newfoundland & Labrador, Canada  JQ422209. officin lis  Foster Island, British Columbia, Canada  KJ637652. officinalis  Somerset, England  JQ917413C. sp. 1 chile  Curinaco, Chile  PTM862C. sp. 1 chile  Curinaco, Chile  PTM863C. sp. 1 chile  Curinaco, Chile  PTM868C. sp. 1 chile  Curinaco, Chile  PTM869C. sp. 1 chile  Curinaco, Chile  PTM876C. sp. 1 chile  Curinaco, Chile  PTM879C. sp. 1 chile  Bonifacio, Chile  PTM889C. sp. 1 chile  Bonifacio, Chile  PTM898C. sp. 1 chile  ar Brava, Chile  PTM910C. sp. 1 chile  Cucao, Chile  PTM926C. sp. 1 chile  Bonifacio, Chile  PTM891C. sp. 1 chile  Bonifacio, Chile  PTM899C. ferreyrae-like  Cape Tachimachi, Hakodate, Japan  PTM1440C. ferreyrae-like  Muroran, Japan  PTM1416C. ferreyr e-like  uroran, Japan  PTM1417C. ferreyrae  Quintay, Chile  PTM819C. ferreyrae  Quintay, Chile  PTM821C. ferreyrae  Quintay, Chile  PTM826C. ferreyrae  Quintay, Chile  PTM827C. ferreyrae  Quintay, Chile  PTM830C. ferreyrae  Quintay, Chile  PTM832C. ferreyrae  Quintay, Chile  PTM833C. ferreyrae  Valparaiso, Chile  PTM847*C. ferreyrae  isotype Pucusana, Peru  MK408748C. sp. 2 chile  Curinaco, Chile  PTM867C. sp. 2 chile  Curinaco, Chile  PTM870C. sp. 2 chile  Curinaco, Chile  PTM873C. sp. 2 chile  Curinaco, Chile  PTM880C. sp. 2 chile  Bonifacio, Chile  PTM895C. sp. 2 c ile  Mar Brava, Chile  PTM905C. sp. 2 chile  Keelung, Taiwan  PTM1519C. sp. 2 chile  California, United States  PTM1254C. sp. 2 chile  California, United States  PTM1262C. sp. 2 chile  California, United States  PTM1265C. sp. 2 chile  California, United States  PTM1266100/99.9193.9/98190/981-/-.96-/-.8681.8/84.8689.4/55.97 97.3/98196.9/88185.8/99175.5/28.7596.1/97184.9/57.8694.5/73190/92.9777.3/5.6492.3/95186.1/22.9595.1/100187.5/43.9974.5/39.7695.9/84144.2/58.93Figure 11psbA treeGenus CorallinaaLRT & 1000 ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian  37    Figure 12. Entire phylogenetic tree of the Corallina genus consisting of 63 CO1 sequences including six outgroups. The top two branch support values are aLRT/Maximum Likelihood percentages (1000 bootstraps). The bottom number is the Bayesian posterior probability. The blue box indicates the portion of the tree that is expanded in Fig. 13. Asterisks denote type sequences.   0.04sp3Japan_PTM1400OUTBfrondUBCa90727KT782032h15sp4Canada_GWS010351JQ615787hs13caespitChile_PTM819CvanCanada_PTM767declinJapan_GWS013767JQ615613HS131chile_Chile_PTM13251chile_Chile_PTM889sp1_OregonPTM12441chile_Chile_PTM8631caliCanada_PTM3631caliUSA_GWS021316JQ615736hs132chile_Chile_PTM870sp4Chile_PTM8441chile_Chile_PTM926likecaespit_Japan_PTM1416sp4Chile_PTM881sp5korea_Korea_GWS018201JQ615795hs131gwsJapan_PTM1457OUTCcheilo_KM254472_Saund1014sp3Canada_GWS006466JQ615765hs13ferreyPeru_MK408747bust19aberransJapanGWS013777JQ615597HS2013crassJapanGWS013776JQ615605HS13CoffCanada_GWS006989JQ615681hs13maxSyntypeJapan_GWS013782JQ615680hs13aberransJapanPTM1445likecaespit_Japan_PTM1440sp3Japan_GWS011941JQ615766hs13likecaespit_PTM1408sp4Chile_PTM8461gwsJapan_GWS013769JQ615738hs13sp2Canada_PTM1178OUTChiBodeg_JQ615596hs132chileChile_PTM895OUTlithoHM918805Hind18co1crassJapan_PTM1447sp1_Canada_PTM332sp3Japan_PTM14422chile_calif_PTM1254CvanCanada_PTM760caespitChile_PTM826CoffepitypeBrodie13BM001062598FM180073caespitHaloBM000804549DQ191343CO1Walker092vanc_Canada_GWS009913JQ615760hs131caliUSA_PTM1247CvanCanada_GWS010831JQ615834hs13like1GWS_Japan_PTM14011caliCanada_PTM1188OUTcrustimur_UBCa89963KU983192h16sp5canada_GWS006561JQ615794hs13sp5Canada_GWS012660HM918986hs13sp1_Canada_PTM182declinJapan_PTM1488OUTellis_GWS001818JQ615843hs13sp4caliGWS021267JQ615770hs132chile_Chile_PTM905sp5Canada_PTM4201chileChile_PTM910crassJapan_PTM1490sp3japan_PTM1405sp2Canada_GWS003062JQ615748hs13likesp3_PTM1439Lithothamnion glaciale  Newf undland & Labrador, Canada HM918805 Ellisolandia elongata  Leitrim, Ireland JQ615843 Calliart ron cheilosporioides  California, United States KM254472Bossiella frondifera  Bamfield, British Columbia, Canada KT782032Chiharaea bodegensis  Bamfield, British Columbia, Canada JQ615596Crusticorallina muricata  Bamfield, British Columbia, Canada KU983192C. maxima Chiba-ken, Japan  JQ615680C. sp. 1 c il   Curinaco, Chile  PTM863C. sp. 1 c il   Cucao, Chile  PTM926C. sp. 1 chile  Cucao, Chile  PTM1325C. sp. 1 chil   Bonifacio, Chile  PTM889C. sp. 1 chile  Mar Brava, Chile  PTM910C. sp. 2 hile  California, United States  PTM1254*C. caespit sa holotype Devon, England  DQ191343C. sp. 2 chile  Bonifacio, Chile  PTM895C. sp. 2 c il   Curinaco, Chile  PTM870C. sp. 2 chile  Mar Brava, Chile  PTM905C. f rreyrae  Quintay, Chile  PTM819C. ferr yrae-like  Cape Tachimachi, Hakodate, Japan PTM1440*C. ferr yrae isotype Pucusana, Peru  MK408747C. ferr yrae-like  Muroran, Japan  PTM1416C. ferr yrae-like Marine Station, Oshoro Bay, Japan PTM1408C. sp. 1 gws-like  Marine Station, Oshoro Bay, Japan  PTM1401C. sp. 1 gws  Chiba-ken, Japan  JQ615738C. sp. 1 gws  Katsuura, Japan  PTM1457C. sp. 2 frondescens  Bamfield, British Columbia, Canada  JQ615748C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM363C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california  California, United States  JQ615736C. sp. 1 california  California, United States  PTM1247C. sp. 5 frondescens  Tahsis, British Columbia, Canada  JQ615794C. sp. 5 frondescens  Haida Gwaii, British Columbia, Canada  HM918986C. sp. 5 frondescens  Hakai, British Columbia, Canada  PTM420C. sp. 4 frondescens  California, United States  JQ615770C. sp. 4 frondescens  Comox, British Columbia, Canada  JQ615787C. ferreyra   Quintay, Chile  PTM826C. sp. 4 frondescens  Curinaco, Chile  PTM881C. sp. 4 frondescens  Valparaiso, Chile  PTM844C. sp. 4 frondescens  Valparaiso, Chile  PTM846C. chilensis Port Renfrew, British Columbia, Canada  PTM182C. chilensis Hakai, British Columbia, Canada  PTM332C. chilensis Oregon, United States  PTM1244C. sp. 5 korea  Piyangdo Island, South Korea  JQ615795C. crassissima  Chiba-ken, Japan  PTM1490C. crassissima  Katsuura, Japan  PTM1447C. crassissima  Chiba-ken, Japan  JQ615605C. declinata Chiba-ken, Japan  JQ615613C. declinata Chiba-ken, Japan  PTM1488C. aberrans Chiba-ken, Japan  JQ61 597C. aberrans Katsuura, Japan  PTM1445. o fici lis  Newfoundland & Labrador, Canada  JQ615681*C. officinalis epitype Devon, England  FM180073C. sp. 2 v couveriensis  British Columbia, Canada  JQ615760. vancouveriensis  British Columbia, Canada  PTM760. vancouveriensis  British Columbia, Canada  JQ615834. vancouveriensis  British Columbia, Canada  PTM767C. sp.  frondescens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Stephenson Point, British Columbia, Canada  JQ615765C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 3 frondescens  Hokkaido, Japan  JQ615766CO1 treeGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian91.6/79175.8/70.8293.9/74198.7/78191.8/87195.6/99197.3/85199.3/65177.7/54.9684.4/93193.4/87198.6/100194.7/80199.5/9910/100.9998.3/891100/100192.9/100180.8/52.76 98.8/98199.4/96195.8/81198.1/100171.2/47.9897.1/100199.7/100181.8/100197.5/98187.9/95.9974.1/34.83100/991 -/-.96 96.1/961Expanded in Fig. 13  38   Figure 13. Expanded portion of the CO1 tree from Fig. 12.  0.04sp3Japan_PTM1400OUTBfrondUBCa90727KT782032h15sp4Canada_GWS010351JQ615787hs13caespitChile_PTM819CvanCanada_PTM767declinJapan_GWS013767JQ615613HS131chile_Chile_PTM13251chile_Chile_PTM889sp1_OregonPTM12441chile_Chile_PTM8631caliCanada_PTM3631caliUSA_GWS021316JQ615736hs132chile_Chile_PTM870sp4Chile_PTM8441chile_Chile_PTM926likecaespit_Japan_PTM1416sp4Chile_PTM881sp5korea_Korea_GWS018201JQ615795hs131gwsJapan_PTM1457OUTCcheilo_KM254472_Saund1014sp3Canada_GWS006466JQ615765hs13ferreyPeru_MK408747bust19aberransJapanGWS013777JQ615597HS2013crassJapanGWS013776JQ615605HS13CoffCanada_GWS006989JQ615681hs13maxSyntypeJapan_GWS013782JQ615680hs13aberransJapanPTM1445likecaespit_Japan_PTM1440sp3Japan_GWS011941JQ615766hs13likecaespit_PTM1408sp4Chile_PTM8461gwsJapan_GWS013769JQ615738hs13sp2Canada_PTM1178OUTChiBodeg_JQ615596hs132chileChile_PTM895OUTlithoHM918805Hind18co1crassJapan_PTM1447sp1_Canada_PTM332sp3Japan_PTM14422chile_calif_PTM1254CvanCanada_PTM760caespitChile_PTM826CoffepitypeBrodie13BM001062598FM180073caespitHaloBM000804549DQ191343CO1Walker092vanc_Canada_GWS009913JQ615760hs131caliUSA_PTM1247CvanCanada_GWS010831JQ615834hs13like1GWS_Japan_PTM14011caliCanada_PTM1188OUTcrustimur_UBCa89963KU983192h16sp5canada_GWS006561JQ615794hs13sp5Canada_GWS012660HM918986hs13sp1_Canada_PTM182declinJapan_PTM1488OUTellis_GWS001818JQ615843hs13sp4caliGWS021267JQ615770hs132chile_Chile_PTM905sp5Canada_PTM4201chileChile_PTM910crassJapan_PTM1490sp3japan_PTM1405sp2Canada_GWS003062JQ615748hs13likesp3_PTM1439Lithothamnion glaciale  Newf undland & Labrador, Canada HM918805 Ellisolandia elongata  Leitrim, Ireland JQ615843 Calliart ron cheilosporioides  California, United States KM254472Bossiella frondifera  Bamfield, British Columbia, Canada KT782032Chiharaea bodegensis  Bamfield, British Columbia, Canada JQ615596Crusticorallina muricata  Bamfield, British Columbia, Canada KU983192C. maxima Chiba-ken, Japan  JQ615680C. sp. 1 c il   Curinaco, Chile  PTM863C. sp. 1 c il   Cucao, Chile  PTM926C. sp. 1 chile  Cucao, Chile  PTM1325C. sp. 1 chil   Bonifacio, Chile  PTM889C. sp. 1 chile  Mar Brava, Chile  PTM910C. sp. 2 hile  California, United States  PTM1254*C. caespit sa holotype Devon, England  DQ191343C. sp. 2 chile  Bonifacio, Chile  PTM895C. sp. 2 c il   Curinaco, Chile  PTM870C. sp. 2 chile  Mar Brava, Chile  PTM905C. f rreyrae  Quintay, Chile  PTM819C. ferr yrae-like  Cape Tachimachi, Hakodate, Japan PTM1440*C. ferr yrae isotype Pucusana, Peru  MK408747C. ferr yrae-like  Muroran, Japan  PTM1416C. ferr yrae-like Marine Station, Oshoro Bay, Japan PTM1408C. sp. 1 gws-like  Marine Station, Oshoro Bay, Japan  PTM1401C. sp. 1 gws  Chiba-ken, Japan  JQ615738C. sp. 1 gws  Katsuura, Japan  PTM1457C. sp. 2 frondescens  Bamfield, British Columbia, Canada  JQ615748C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM363C. sp. 1 c lifornia  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california  California, United States  JQ615736C. sp. 1 california  California, United States  PTM1247C. sp. 5 frondescens  Tahsis, British Columbia, Canada  JQ615794C. sp. 5 frondescens  Haida Gwaii, British Columbia, Canada  HM918986C. sp. 5 frondescens  Hakai, British Columbia, Canada  PTM420C. sp. 4 frondescens  California, United States  JQ615770C. sp. 4 frondescens  Comox, British Columbia, Canada  JQ615787C. ferreyra   Quintay, Chile  PTM826C. sp. 4 frondescens  Curinaco, Chile  PTM881C. sp. 4 frondescens  Valparaiso, Chile  PTM844C. sp. 4 frondescens  Valparaiso, Chile  PTM846C. chilensis Port Renfrew, British Columbia, Canada  PTM182C. chilensis Hakai, British Columbia, Canada  PTM332C. chilensis Oregon, United States  PTM1244C. sp. 5 korea  Piyangdo Island, South Korea  JQ615795C. crassissima  Chiba-ken, Japan  PTM1490C. crassissima  Katsuura, Japan  PTM1447C. crassissima  Chiba-ken, Japan  JQ615605C. declinata Chiba-ken, Japan  JQ615613C. declinata Chiba-ken, Japan  PTM1488C. aberrans Chiba-ken, Japan  JQ61 597C. aberrans Katsuura, Japan  PTM1445. o fici lis  Newfoundland & Labrador, Canada  JQ615681*C. officinalis epitype Devon, England  FM180073C. sp. 2 v couveriensis  British Columbia, Canada  JQ615760. vancouveriensis  British Columbia, Canada  PTM760. vancouveriensis  British Columbia, Canada  JQ615834. vancouveriensis  British Columbia, Canada  PTM767C. sp.  frondescens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. sp. 3 frondescens  Stephenson Point, British Columbia, Canada  JQ615765C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 3 frondescens  Hokkaido, Japan  JQ615766CO1 treeGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian91.6/79175.8/70.8293.9/74198.7/78191.8/87195.6/99197.3/85199.3/65177.7/54.9684.4/93193.4/87198.6/100194.7/80199.5/9910/100.9998.3/891100/100192.9/100180.8/52.76 98.8/98199.4/96195.8/81198.1/100171.2/47.9897.1/100199.7/100181.8/100197.5/98187.9/95.9974.1/34.83100/991 -/-.96 96.1/961Expanded in Fig. 13  39     Figure 14. Entire phylogenetic tree of 47 rbcL sequences of Corallina and six outgroups. Sequences from the herbarium materials from the 1800’s are not included in this tree. The top two branch support values are aLRT/Maximum Likelihood percentages (1000 bootstraps); the bottom number is the Bayesian posterior probability. The blue box indicates the portion of the tree that is expanded in Fig. 15. Asterisks denote type sequences.   rbcL0.04likecaespit_Japan_PTM1416melobesJapan_UBC_A62034JN701477Martone12Cpinntopotype_USAA88590HQ322333Mart12crassJapan_PTM1447CoffCanada_NCU590595KJ591674h14OUTchiharbod_JQ677000hs13otherCoff_BM001004107JN701476h141cali_Canada_PTM11882chile_Chile_PTM870sp2_Canada_PTM1178sp3_Japan_PTM14051GWS_Japan_PTM14571cali_USA_PTM1247like1gws_Japan_PTM1409sp5Canada_PTM420sp1_Canada_PTM332ferreyrae1chileChile_PTM1325like1GWS_Japan_PTM1401likecaespit_Japan_PTM1408CvanCanada_PTM767sp4_Chile_PTM844likesp3_Japan_PTM1419decabe_Japan_PTM1445crassJapan_PTM_1490sp1_Oregon_PTM1244caespitChile_PTM8262chile_USA_PTM1254OUTellis_BM000806006KP834400will15OUTcrusticurcat_UBCa89963KU983253h16Coffepitype_BM001062598JX315329Brod131chile_Chile_PTM8991chile_Chile_PTM863sp3__Japan_PTM1400sp3_Japan_PTM1442sp4_Oregon_PTM12351cali_Canada_PTM515OUTlithoglas_KC134336h18_rbcLlike1GWS_Japan_PTM1402OUTcallicheilo_HQ322294Gabr11likesp3_Japan_PTM1439CoffAlaska_NCU588445KJ591672h14likecaespit_Japan_PTM1440OUTBfrondif_UBCa90727KT782137h151cali_Canada_PTM3631chile_Chile_PTM9101chile_Chile_PTM1337Lithothamnion glaciale  Newfoundland & Labrador, Canada KC134336 Elliso andia elongata  Llanes, Asturias, Spain KP834400 Calliarthron cheilosporioides  California, United States HQ322294Bossiella frondifera  Bamfield, British Columbia Canada KT782137Chiharaea bodegensis  Canadian Northeast Pacific?  JQ677000Crusticorallina muric ta  Botany Bay, British Columbia, Canada KU983253C. sp. 3 frondescens-like  Cape Tachimachi, Hakodate, Japan  PTM1439C. sp. 3 frondescens-like  Muroran, Japan  PTM1419C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. officinalis  Alaska, United States  KJ591672. officinalis Foster Island British Columbia, Canada  KJ591674. officinalis Somerset, England  JN701476*C. Officinalis epitype Devon, England  JX315329. vancouveriensis  Hakai, British Columbia, Canada  PTM767C. sp. 1 california  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california Hakai, British Columbia, Canada  PTM363C. sp. 1 california  Hakai, British Columbia, Canada  PTM515C. sp. 1 california  California, United States  PTM1247C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. chile sis Hakai, British Columbia, Canada  PTM332C. chilensis  Oregon, United States  PTM1244C. aberrans Katsuura, Japan  PTM1445C. crassissima  Chiba-ken, Japan  PTM1490C. crassissima arine Institute, Katsuura, Japan  PTM1447C. sp. 5 frondescens Hakai, British Columbia, Canada  PTM420C. sp. 4 fr descens Oregon, United States  PTM1235C. sp. 4 frondescens Valparaiso, Chile  PTM844C. sp. 1 gws Katsuura, Japan  PTM1457C. sp. 1 gws-like Marine Station, Oshoro Bay, Japan  PTM1409C. sp. 1 gws-like Marine Station, Oshoro Bay, Japan  PTM1401C. sp. 1 gws-like Marine Stations, Oshoro Bay, Japan  PTM1402C. ferreyra Quintay, Chile  PTM826*C. ferreyrae isotype Pucusana, Peru  C. ferr yrae-like Cape Tachimachi, Hakodate, Japan  PTM1440C. ferr yrae-like Muroran, Japan  PTM1416C. ferr yrae-like Marine Station, Oshoro Bay, Japan  PTM1408C. sp. 2 chile Curinaco, Chile  PTM870C. melobesioides Awa-Kominato, Chiba-ke , Japan  JN701477. pi natifolia California, United States  HQ322333C. sp. 2 chile California, United States  PTM1254C. sp. 1 chile Mar Brava, Chile  PTM910C. sp. 1 chile Bonifacio, Chile  PTM899C. sp. 1 chile Cucao, Chile  PTM1325C. sp. 1 chile Curinaco, Chile  PTM863C. sp. 1 chile Pucatrihue, Chile  PTM1337Figure 14rbcL treeGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian96.3/99114.3/55.786.5/84.9891/80193.1/75193.6/87194.4/99186.8/92197.8/95196.6/99188/66186.6/59191.8/93196.2/991100/99189.7/100177.5/27.7277.3/68.85 99.8/100199.9/98195.7/100189.1/95182/38.8199.5/100199.7/100178.6/47.8230/64.8885.6/83.9899.7/100.88Expanded in Fig. 15  40  Figure 15. Expanded portion of the rbcL tree from Fig. 14.  rbcL0.04likecaespit_Japan_PTM1416melobesJapan_UBC_A62034JN701477Martone12Cpinntopotype_USAA88590HQ322333Mart12crassJapan_PTM1447CoffCanada_NCU590595KJ591674h14OUTchiharbod_JQ677000hs13otherCoff_BM001004107JN701476h141cali_Canada_PTM11882chile_Chile_PTM870sp2_Canada_PTM1178sp3_Japan_PTM14051GWS_Japan_PTM14571cali_USA_PTM1247like1gws_Japan_PTM1409sp5Canada_PTM420sp1_Canada_PTM332ferreyrae1chileChile_PTM1325like1GWS_Japan_PTM1401likecaespit_Japan_PTM1408CvanCanada_PTM767sp4_Chile_PTM844likesp3_Japan_PTM1419decabe_Japan_PTM1445crassJapan_PTM_1490sp1_Oregon_PTM1244caespitChile_PTM8262chile_USA_PTM1254OUTellis_BM000806006KP834400will15OUTcrusticurcat_UBCa89963KU983253h16Coffepitype_BM001062598JX315329Brod131chile_Chile_PTM8991chile_Chile_PTM863sp3__Japan_PTM1400sp3_Japan_PTM1442sp4_Oregon_PTM12351cali_Canada_PTM515OUTlithoglas_KC134336h18_rbcLlike1GWS_Japan_PTM1402OUTcallicheilo_HQ322294Gabr11likesp3_Japan_PTM1439CoffAlaska_NCU588445KJ591672h14likecaespit_Japan_PTM1440OUTBfrondif_UBCa90727KT782137h151cali_Canada_PTM3631chile_Chile_PTM9101chile_Chile_PTM1337Lithothamnion glaciale  Newfoundland & Labrador, Canada KC134336 Elliso andia elongata  Llanes, Asturias, Spain KP834400 Calliarthron cheilosporioides  California, United States HQ322294Bossiella frondifera  Bamfield, British Columbia Canada KT782137Chiharaea bodegensis  Canadian Northeast Pacific?  JQ677000Crusticorallina muric ta  Botany Bay, British Columbia, Canada KU983253C. like sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1439C. like sp. 3 frondescens  Muroran, Japan  PTM1419C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1400C. sp. 3 frondescens  Cape Tachimachi, Hakodate, Japan  PTM1442C. sp. 3 frondescens  Marine Station, Oshoro Bay, Japan  PTM1405C. officinalis  Alaska, United States  KJ591672. officinalis Foster Island British Columbia, Canada  KJ591674. officinalis Somerset, England  JN701476*C. Officinalis epitype Devon, England  JX315329. vancouveriensis  Hakai, British Columbia, Canada  PTM767C. sp. 1 california  Hakai, British Columbia, Canada  PTM1188C. sp. 1 california Hakai, British Columbia, Canada  PTM363C. sp. 1 california  Hakai, British Columbia, Canada  PTM515C. sp. 1 california  California, United States  PTM1247C. sp. 2 frondescens  Hakai, British Columbia, Canada  PTM1178C. chile sis Hakai, British Columbia, Canada  PTM332C. chilensis  Oregon, United States  PTM1244C. aberrans  Katsuura, Japan  PTM1445C. crassissima  Chiba-ken, Japan  PTM1490C. crassissima arine Institute, Katsuura, Japan  PTM1447C. sp. 5 frondescens Hakai, British Columbia, Canada  PTM420C. sp. 4 fr descens Oregon, United States  PTM1235C. sp. 4 frondescens Valparaiso, Chile  PTM844C. sp. 1 gws Katsuura, Japan  PTM1457C. like sp. 1 gws Marine Station, Oshoro Bay, Japan  PTM1409C. like sp. 1 gws Marine Station, Oshoro Bay, Japan  PTM1401C. like sp. 1 gws Marine Stations, Oshoro Bay, Japan  PTM1402C. ferreyra Quintay, Chile  PTM826*C. ferreyrae isotype Pucusana, Peru  C. like ferreyrae Cape Tachimachi, Hakodate, Japan  PTM1440C. like ferreyrae Muroran, Japan  PTM1416C. like ferreyrae Marine Station, Oshoro Bay, Japan  PTM1408C. sp. 2 chile Curinaco, Chile  PTM870C. melobesioides Awa-Kominato, Chiba-ke , Japan  JN701477. pi natifolia California, United States  HQ322333C. sp. 2 chile California, United States  PTM1254C. sp. 1 chile Mar Brava, Chile  PTM910C. sp. 1 chile Bonifacio, Chile  PTM899C. sp. 1 chile Cucao, Chile  PTM1325C. sp. 1 chile Curinaco, Chile  PTM863C. sp. 1 chile Pucatrihue, Chile  PTM1337Figure 15rbcL treeGenus CorallinaaLRT & 1000 bootstrap ML percentagesBayesian posterior probabilitiesaLRT/MLBayesian96.3/99114.3/55.786.5/84.9891/80193.1/75193.6/87194.4/99186.8/92197.8/95196.6/99188/66186.6/59191.8/93196.2/991100/99189.7/100177.5/27.7277.3/68.85 99.8/100199.9/98195.7/100189.1/95182/38.8199.5/100199.7/100178.6/47.8230/64.8885.6/83.9899.7/100.88  41    Figure 16. This Corallina 50% majority rule tree shows species-level clades that are shared across separate bootstrap consensus trees from psbA, CO1, and rbcL gene sequences. Percentages indicate whether the group was present in one (33%), two (67%), or three (100%) of the individual gene trees. The polytomies and instances of 33% support are due to conflict across loci, to limitations to the phylogenetic resolution possible from each locus, and to missing data from some loci for some taxa. Short or nonsense branches are relics of having had no data associated with taxa during intermediary steps in the analysis and should be disregarded. Relationships among species are not resolved consistently across the three loci.   40.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_123This is a “majority-rule” consensus tree of psbA, CO1, and rbcL genes for the Corallina genus.  It is NOT concatenated, but demonstrates the disagreementbetween genes, (which is why it is inappropriate to concatenate).This tree is a majority-rule >50 tree created from three other fundamental treeswhich were also created via majority rule >50 analyses.The first three fundamental trees were created by running 1,000 bootstraps on each individual gene tree.  Then a majority rule consensus tree was created for each gene using the 1,000 tree versions.  After that a majority rule analysis was run on the three fundamental trees simultaneously to create what you see here.  The numerical values represent the frequency of clades from the three fundamental trees.  For example, if you see “33”, it only occurredin that particular arrangement in one of the genes.  If you see “67,” two of the gene trees agreed.  Lithothamnion was the outermost outgroup.Taxon naming key:Cchile_740_psbA means “C. chilensis_PTM# 740_only psbA gene”GWS_1470_123 means “C. sp. 1 GWS_PTM# 1470_all three genes (123)“123” = psbA, CO1, rbcL“1X3” – yes psbA, NO CO1, yes rbcL“X23” = NO psbA, yes CO1, yes rbcL“OUT” = “out group”Example:  Cchile_JN1234_psbA = published sequence w/ genbank#Genus CorallinaMajority rule consensus333333333333333333333333333333333333333333333367100676767C. chilensis  42    Figure 17. Expanded from Fig. 16.    40.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_123C. chilensisC. sp. 5 frondescensC. sp. 1 californiaC. sp. 4 frondescensC. sp. 1 chileC. sp. 1 gws & gws-like333333333333333333333333333340.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_123  43      Figure 18. Expanded from Fig. 16.    40.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_12340.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_12333 C. sp. 2 chileC. ferreyraeC. officinalis psbAC. sp. 2 frondescensC. vancouveriensis & C. sp. 2 vancouveriensisC. sp. 3 frondescensC. sp. 3 frondescens-like333333333333333333336733  44      Figure 19. Expanded from Fig. 16.    40.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_123676740.0sp4fro_JQ422222_psbA1chile_879_psbAsp3fro_JQ422221_psbACoff_KJ637652_psbACoffEpi_FM180073_CO1Cvan_JQ615834_CO11chile_876_psbAferreylike_1417_psbAcali_363_1232chile_1254_123ferreylike_1408_X23Cvan_767_123aberr_JQ422201_psbAsp3like_1419_1X3sp5fro_JQ422227_psbACoff_JQ615681_CO1GWSlike_1409_rbcLferrey_bust_1231chile_862_psbACoff_KJ637651_psbAsp2fro_1178_1231chile_891_psbA2chile_873_psbA2chile_895_12Xsp4fro_846_12Xferrey_819_12XCvan_760_12XcaesHolo_DQ191343_CO1Cchil_740_psbAferrey_827_psbAsp3fro_JQ615766_CO1OUTBfrond_123sp3fro_JQ615765_CO1Coff_Jn701476_rbcLsp4fro_1235_1X3ferrey_830_psbACchil_738_psbAsp3fro_1400_123Cchil_742_psbAOUTLithotham_123sp3fro_1442_123Cchil_332_123Cvan_JQ422228_psbA2chile_1265_psbA2chile_1519_psbAsp4fro_881_12X1chile_863_1232chile_1266_psbA1chile_926_12XCoff_JQ917413_psbAsp2fro_489_psbA1chile_899_1X3CoffEpi_JX315329_rbcLOUTCrusti_123sp5fro_420_123ferrey_833_psbACchil_1244_1232chile_867_psbAsp4fro_822_psbAaberr_1445_123ferreylike_1416_1232chile_870_123cali_JQ422238_psbAcali_515_1X31chile_910_123Coff_JQ422209_psbACoff_KJ591672_rbcLferrey_821_psbAcali_1247_1231chile_889_12Xsp4fro_842_psbACchil_789_psbAdeclin_JQ422204_psbAspKorea_JQ615795_CO1declin_JQ615613_CO1maxSyn_JQ615680_CO1sp5fro_JQ615794_CO11chile_869_psbAferreylike_1440_123crass_1447_X23sp5fro_HM918986_CO1ferrey_832_psbACchil_182_12X1chile_898_psbAmaxSyn_JQ422207_psbAaberr_JQ615597_CO1sp4fro_JQ615770_CO1sp3fro_1405_psbA_123sp3like_1439_123OUTChihar_123GWSlike_1402_rbcL2chile_1262_psbAcrass_JQ615605_CO1ferrey_847_psbAsp4fro_JQ615787_CO1GWS_JQ615738_CO1sp2fro_JQ615748_CO1Coff_KJ591674_rbcLCvan2_JQ422229_psbAsp5fro_JQ422226_psbAcrass_JQ422203_psbA2chile_905_12X1chile_1337_rbcLOUTEllis_123melobes_Jn701477_rbcL1chile_868_psbACvan2_JQ615760_CO1sp4fro_844_123Cchil_743_psbAcrass_1490_123GWS_JQ422217_psbAcali_JQ6157_CO1GWSlike_1401_123Cchil_788_psbAOUTCalliar_1231chile_1325_X23GWS_1457_123pinnTopo_HQ322333_rbcLdeclin_1488_12X2chile_880_psbAcali_1188_12367  45    Figure 20. Indicating phylogenetic conflict among loci, individual gene sequences of C. officinalis appear in three locations in a maximum likelihood tree in which other taxa are represented by concatenated psbA/CO1/rbcL sequences. Numbers are bootstrap percentages. Outgroups are concatenated as specified in Table S3, Appendix II.     Snake tree0.2crass_JQ615605_CO1ferrey_827_psbA1chile_1325_X23ferrey_833_psbAmaxSyn_JQ422207_psbAdeclin_1488_12Xferrey_821_psbACchil_789_psbAsp5fro_JQ422227_psbACoffEpi_FM180073_CO1sp5fro_420_123cali_1188_123cali_JQ6157_CO11chile_910_123Cvan_JQ615834_CO1OUTLithotham_1232chile_1266_psbA1chile_898_psbACvan_767_123GWSlike_1402_rbcLferrey_847_psbACoff_KJ591674_rbcLcali_363_123sp4fro_822_psbAsp2fro_1178_123Cchil_182_12XOUTBfrond_123GWSlike_1409_rbcLOUTCalliar_1232chile_1262_psbAsp5fro_JQ615794_CO1Cchil_788_psbACchil_742_psbAsp3fro_1405_psbA_123Cvan2_JQ422229_psbA2chile_1519_psbACoff_KJ637652_psbACchil_743_psbA2chile_880_psbAOUTCrusti_123sp4fro_846_12XCvan_JQ422228_psbAsp4fro_JQ615787_CO1sp4fro_844_123pinnTopo_HQ322333_rbcLCchil_1244_123cali_515_1X3sp3fro_JQ615765_CO12chile_1265_psbAferrey_bust_1231chile_1337_rbcLcali_JQ422238_psbACvan2_JQ615760_CO1caesHolo_DQ191343_CO11chile_899_1X3sp4fro_JQ615770_CO12chile_895_12XCchil_740_psbAferrey_819_12Xaberr_JQ615597_CO1ferreylike_1408_X231chile_926_12X1chile_879_psbAsp3like_1439_123cali_1247_123sp4fro_842_psbAsp3fro_1400_123sp3fro_JQ615766_CO1declin_JQ422204_psbA2chile_867_psbA2chile_905_12X1chile_876_psbA1chile_891_psbACoff_Jn701476_rbcLcrass_1490_123Cchil_332_123melobes_Jn701477_rbcLferrey_832_psbAferreylike_1440_1231chile_868_psbAspKorea_JQ615795_CO11chile_863_123Coff_JQ422209_psbAGWSlike_1401_123ferreylike_1417_psbAcrass_1447_X23sp3fro_JQ422221_psbAsp3fro_1442_123OUTEllis_1231chile_862_psbAferreylike_1416_123GWS_1457_123Coff_JQ917413_psbAsp4fro_1235_1X3sp2fro_489_psbAsp5fro_JQ422226_psbAsp3like_1419_1X32chile_873_psbAferrey_830_psbA2chile_870_123sp5fro_HM918986_CO1aberr_1445_123sp4fro_JQ422222_psbAOUTChihar_123Coff_KJ591672_rbcL1chile_869_psbAcrass_JQ422203_psbAGWS_JQ422217_psbAsp2fro_JQ615748_CO1Cchil_738_psbAGWS_JQ615738_CO1sp4fro_881_12XmaxSyn_JQ615680_CO1Coff_KJ637651_psbAdeclin_JQ615613_CO1aberr_JQ422201_psbA1chile_889_12XCvan_760_12X2chile_1254_123Coff_JQ615681_CO1CoffEpi_JX315329_rbcL7097100863987384619842873699919410010025349910073883868999799976499699873375989388509510010039utgr up:  Lithothamnion glaciale, concatenatedutgroup:  Calliarthron cheilosporioides, concatenatedutgroup:  Chiharaea bodegensis, concatenatedutgroup:  Bossiella frondifera, concatenatedOutgroup:  Ellisolandia elongata, concatenatedutgroup:  Crusticorallina muricata, concatenatedC. chilensis (N=9)C. sp. 1 california (N=6)C. sp. 4 frondescens (N=9)C. sp. 5 frondescens (N=5)C. sp. 2 frondescens (N=3)C. officinalis psbA (N=4)C. sp. 3 frondescens-like (N=2)C. officinalis rbcL (N=6)C. officinalis CO1 (N=2)C. sp. 3 frondescens (N=6)C. vancouveriensis (N=4)C. sp. 2 vancouveriensis (N=2)C. sp. 1 chile (N=14)C. ferreyrae (N=8)C. ferreyrae-like (N=4)C. sp. 2 chile complexC. crassissima complexC. sp. 1 gws (N=3)C. sp. 1 gws-like (N=3)C. maxima (N=2)Sequence naming key:• species_collection#_gene(s)• PTM# unless stated otherwise• “123” = psbA, CO1 & rbcL• “12X” = psbA & CO1• “1X3” = psbA & rbcL• “X23” = CO1 & rbcL• “psbA” = only psbAExpansion 1Expansion 2  46    Figure 21. Expanded from Fig. 20.  Snake tree0.2crass_JQ615605_CO1ferrey_827_psbA1chile_1325_X23ferrey_833_psbAmaxSyn_JQ422207_psbAdeclin_1488_12Xferrey_821_psbACchil_789_psbAsp5fro_JQ422227_psbACoffEpi_FM180073_CO1sp5fro_420_123cali_1188_123cali_JQ6157_CO11chile_910_123Cvan_JQ615834_CO1OUTLithotham_1232chile_1266_psbA1chile_898_psbACvan_767_123GWSlike_1402_rbcLferrey_847_psbACoff_KJ591674_rbcLcali_363_123sp4fro_822_psbAsp2fro_1178_123Cchil_182_12XOUTBfrond_123GWSlike_1409_rbcLOUTCalliar_1232chile_1262_psbAsp5fro_JQ615794_CO1Cchil_788_psbACchil_742_psbAsp3fro_1405_psbA_123Cvan2_JQ422229_psbA2chile_1519_psbACoff_KJ637652_psbACchil_743_psbA2chile_880_psbAOUTCrusti_123sp4fro_846_12XCvan_JQ422228_psbAsp4fro_JQ615787_CO1sp4fro_844_123pinnTopo_HQ322333_rbcLCchil_1244_123cali_515_1X3sp3fro_JQ615765_CO12chile_1265_psbAferrey_bust_1231chile_1337_rbcLcali_JQ422238_psbACvan2_JQ615760_CO1caesHolo_DQ191343_CO11chile_899_1X3sp4fro_JQ615770_CO12chile_895_12XCchil_740_psbAferrey_819_12Xaberr_JQ615597_CO1ferreylike_1408_X231chile_926_12X1chile_879_psbAsp3like_1439_123cali_1247_123sp4fro_842_psbAsp3fro_1400_123sp3fro_JQ615766_CO1declin_JQ422204_psbA2chile_867_psbA2chile_905_12X1chile_876_psbA1chile_891_psbACoff_Jn701476_rbcLcrass_1490_123Cchil_332_123melobes_Jn701477_rbcLferrey_832_psbAferreylike_1440_1231chile_868_psbAspKorea_JQ615795_CO11chile_863_123Coff_JQ422209_psbAGWSlike_1401_123ferreylike_1417_psbAcrass_1447_X23sp3fro_JQ422221_psbAsp3fro_1442_123OUTEllis_1231chile_862_psbAferreylike_1416_123GWS_1457_123Coff_JQ917413_psbAsp4fro_1235_1X3sp2fro_489_psbAsp5fro_JQ422226_psbAsp3like_1419_1X32chile_873_psbAferrey_830_psbA2chile_870_123sp5fro_HM918986_CO1aberr_1445_123sp4fro_JQ422222_psbAOUTChihar_123Coff_KJ591672_rbcL1chile_869_psbAcrass_JQ422203_psbAGWS_JQ422217_psbAsp2fro_JQ615748_CO1Cchil_738_psbAGWS_JQ615738_CO1sp4fro_881_12XmaxSyn_JQ615680_CO1Coff_KJ637651_psbAdeclin_JQ615613_CO1aberr_JQ422201_psbA1chile_889_12XCvan_760_12X2chile_1254_123Coff_JQ615681_CO1CoffEpi_JX315329_rbcL7097100863987384619842873699919410010025349910073883868999799976499699873375989388509510010039utgr up:  Lithothamnion glaciale, concatenatedutgroup:  Calliarthron cheilosporioides, concatenatedutgroup:  Chiharaea bodegensis, concatenatedutgroup:  Bossiella frondifera, concatenatedOutgroup:  Ellisolandia elongata, concatenatedutgroup:  Crusticorallina muricata, concatenatedC. chilensis (N=9)C. sp. 1 california (N=6)C. sp. 4 frondescens (N=9)C. sp. 5 frondescens (N=5)C. sp. 2 frondescens (N=3)C. officinalis psbA (N=4)C. sp. 3 frondescens-like (N=2)C. officinalis rbcL (N=6)C. officinalis CO1 (N=2)C. sp. 3 frondescens (N=6)C. vancouveriensis (N=4)C. sp. 2 vancouveriensis (N=2)C. sp. 1 chile (N=14)C. ferreyrae (N=8)C. ferreyrae-like (N=4)C. sp. 2 chile complexC. crassissima complexC. sp. 1 gws (N=3)C. sp. 1 gws-like (N=3)C. maxima (N=2)Sequence naming key:• species_collection#_gene(s)• PTM# unless stated otherwise• “123” = psbA, CO1 & rbcL• “12X” = psbA & CO1• “1X3” = psbA & rbcL• “X23” = CO1 & rbcL• “psbA” = only psbAExpansion 1Expansion 2  47   Figure 22. Expanded from Fig. 20.  Snake tree0.2crass_JQ615605_CO1ferrey_827_psbA1chile_1325_X23ferrey_833_psbAmaxSyn_JQ422207_psbAdeclin_1488_12Xferrey_821_psbACchil_789_psbAsp5fro_JQ422227_psbACoffEpi_FM180073_CO1sp5fro_420_123cali_1188_123cali_JQ6157_CO11chile_910_123Cvan_JQ615834_CO1OUTLithotham_1232chile_1266_psbA1chile_898_psbACvan_767_123GWSlike_1402_rbcLferrey_847_psbACoff_KJ591674_rbcLcali_363_123sp4fro_822_psbAsp2fro_1178_123Cchil_182_12XOUTBfrond_123GWSlike_1409_rbcLOUTCalliar_1232chile_1262_psbAsp5fro_JQ615794_CO1Cchil_788_psbACchil_742_psbAsp3fro_1405_psbA_123Cvan2_JQ422229_psbA2chile_1519_psbACoff_KJ637652_psbACchil_743_psbA2chile_880_psbAOUTCrusti_123sp4fro_846_12XCvan_JQ422228_psbAsp4fro_JQ615787_CO1sp4fro_844_123pinnTopo_HQ322333_rbcLCchil_1244_123cali_515_1X3sp3fro_JQ615765_CO12chile_1265_psbAferrey_bust_1231chile_1337_rbcLcali_JQ422238_psbACvan2_JQ615760_CO1caesHolo_DQ191343_CO11chile_899_1X3sp4fro_JQ615770_CO12chile_895_12XCchil_740_psbAferrey_819_12Xaberr_JQ615597_CO1ferreylike_1408_X231chile_926_12X1chile_879_psbAsp3like_1439_123cali_1247_123sp4fro_842_psbAsp3fro_1400_123sp3fro_JQ615766_CO1declin_JQ422204_psbA2chile_867_psbA2chile_905_12X1chile_876_psbA1chile_891_psbACoff_Jn701476_rbcLcrass_1490_123Cchil_332_123melobes_Jn701477_rbcLferrey_832_psbAferreylike_1440_1231chile_868_psbAspKorea_JQ615795_CO11chile_863_123Coff_JQ422209_psbAGWSlike_1401_123ferreylike_1417_psbAcrass_1447_X23sp3fro_JQ422221_psbAsp3fro_1442_123OUTEllis_1231chile_862_psbAferreylike_1416_123GWS_1457_123Coff_JQ917413_psbAsp4fro_1235_1X3sp2fro_489_psbAsp5fro_JQ422226_psbAsp3like_1419_1X32chile_873_psbAferrey_830_psbA2chile_870_123sp5fro_HM918986_CO1aberr_1445_123sp4fro_JQ422222_psbAOUTChihar_123Coff_KJ591672_rbcL1chile_869_psbAcrass_JQ422203_psbAGWS_JQ422217_psbAsp2fro_JQ615748_CO1Cchil_738_psbAGWS_JQ615738_CO1sp4fro_881_12XmaxSyn_JQ615680_CO1Coff_KJ637651_psbAdeclin_JQ615613_CO1aberr_JQ422201_psbA1chile_889_12XCvan_760_12X2chile_1254_123Coff_JQ615681_CO1CoffEpi_JX315329_rbcL7097100863987384619842873699919410010025349910073883868999799976499699873375989388509510010039utgr up:  Lithothamnion glaciale, concatenatedutgroup:  Calliarthron cheilosporioides, concatenatedutgroup:  Chiharaea bodegensis, concatenatedutgroup:  Bossiella frondifera, concatenatedOutgroup:  Ellisolandia elongata, concatenatedutgroup:  Crusticorallina muricata, concatenatedC. chilensis (N=9)C. sp. 1 california (N=6)C. sp. 4 frondescens (N=9)C. sp. 5 frondescens (N=5)C. sp. 2 frondescens (N=3)C. officinalis psbA (N=4)C. sp. 3 frondescens-like (N=2)C. officinalis rbcL (N=6)C. officinalis CO1 (N=2)C. sp. 3 frondescens (N=6)C. vancouveriensis (N=4)C. sp. 2 vancouveriensis (N=2)C. sp. 1 chile (N=14)C. ferreyrae (N=8)C. ferreyrae-like (N=4)C. sp. 2 chile complexC. crassissima complexC. sp. 1 gws (N=3)C. sp. 1 gws-like (N=3)C. maxima (N=2)Sequence naming key:• species_collection#_gene(s)• PTM# unless stated otherwise• “123” = psbA, CO1 & rbcL• “12X” = psbA & CO1• “1X3” = psbA & rbcL• “X23” = CO1 & rbcL• “psbA” = only psbAExpansion 1Expansion 2  48 1.3.1 Corallina chilensis is not a variety of C. officinalis    If Kützing were correct and Corallina chilensis were a variety of C. officinalis, C. chilensis would have to be monophyletic with C. officinalis in the gene trees (Figs. 10-15). Further, C. officinalis and C. chilensis sequences would be expected to show a high percent similarity in distance matrices (Appendix V), and the two would group together in the barcode gap analyses (Table 2).   In the preliminary ABGD analyses (Table 2), one would expect to see C. chilensis and C. officinalis delimited as separate species consistently across all three genes if C. chilensis were not a variety of C. officinalis. However, C. chilensis and C. officinalis were delimited as separate species with respect to CO1 and rbcL, although they were grouped as the same candidate species along with C. sp. 1 california, C. sp. 2 frondescens, and C. sp. 5 frondescens in the psbA analysis (Table 2).  Corallina chilensis likewise did not fit with the expectations for varietal status in the phylogenetic analyses. The C. chilensis clade was not monophyletic with the C. officinalis clade in any of the gene trees (Figs. 8-22). In the psbA tree, C. chilensis sequences formed a clade with strong branch support that was nested among species other than C. officinalis (Figs. 10-11), which occupied its own strongly supported clade (Figs. 10-11). Corallina chilensis occurred in a clade with C. sp. 1 california and C. sp. 5 frondescens whereas C. officinalis occurred within a clade containing C. sp. 1 chile, C. sp. 2 chile, C. ferreryrae, and C. ferreyrae-like (Figs. 10-11).  In the CO1 tree, C. officinalis sequences formed a clade with 81.8/100/1 branch support (aLRT percent value/bootstrap percent value/Bayesian posterior probability) (Figs. 12-13). Corallina officinalis was sister to C. vancouveriensis (97.5/98/1 branch support) in the CO1 tree   49 rather than sister to C. chilensis (Figs. 12-13). Corallina chilensis formed a clade with C. crassissima, C. declinata, C. aberrans, and C. sp. 5 korea in the CO1 tree (Figs. 12-13).   In the rbcL tree, C. chilensis and C. officinalis formed two separate strongly supported clades although it was difficult to determine their nearest sister relationships due to the polytomies (Figs. 14-15). However, in the concatenated tree (Figs. 20-22), C. officinalis rbcL sequences were sister to the C. sp. 3 frondescens clade. Corallina officinalis psbA sequences formed a clade with C. sp. 2 frondescens, and C. officinalis CO1 sequences were sister to the C. vancouveriensis complex (Figs. 20-22). Corallina officinalis was sister to three different species or species complexes within the concatenated tree, none of which were, or contained, C. chilensis. In the majority-rule tree (Figs. 16-19), C. officinalis did not form a clade, but C. chilensis formed a clade that was not sister to any C. officinalis sequences.   When analyzing raw pairwise distances, one would expect that if C. chilensis were a variety of C. officinalis, the two would demonstrate intraspecific levels of percent difference. Instead, upon analyzing raw pairwise distances, C. chilensis differed from C. officinalis by 0.94-1.06, 6.31-7.26, and 1.8-2.36% across psbA, CO1, and rbcL gene distance matrices respectively, levels consistent with interspecific variation (Table 3, Appendix V). The high percent differences between C. chilensis and C. officinalis across all three genes (Table 3, Appendix V) indicates that the two are distinct species from one another.        50 Table 3. Summary table (derived from full matrices in Appendix V) presenting the range of percent difference between C. chilensis and other known species or potentially closely related species within Corallina.    Overall, lack of monophyly of C. chilensis with C. officinalis, separation of the two in ABGD delimitation, and separation of the two by a consistent pattern of pairwise distances was consistent with expectations under the assumption that they are separate species.  1.3.2 Specimens from the Northern Hemisphere matched the C. chilensis holotype & formed a clade in all trees.   Analysis of single loci and concatenated data from the three loci provided congruent phylogenetic support for a clade of specimens including the C. chilensis holotype collected by Darwin (#2151) in Valparaiso, Chile. The C. chilensis holotype (Fig. 4 and Appendix II, Table S1) occurred in a clade with 97.1/62/- support in the rbcL type tree (Figs. 8-9). The 263 bp rbcL sequence from the holotype was identical over its length to rbcL sequences from what had been referred to as Corallina sp. 1 frondescens (Appendix II, Table S1/PTM 332 UBC A89284, British Columbia, Canada) from the Northern Hemisphere (Figs. 8-9). I therefore refer to C. chilensis C. chilensis C. chilensispsb A CO1 rbc LC. chilensis 0-0.24 0.45-0.91 0.09-0.09C. officinalis 0.94-1.06 6.31-7.26 1.8-2.36C. vancouveriensis 1.06-1.18 7.72-8.62 2.08-2.18C. crassissima 1.29-1.41 6.2-6.66 1.95-2.08C. ferreyrae 0.94-1.06 8.62-9.08 1.72-1.9C. sp. 2 frondescens 0.59-0.82 6.96-7.26 1.12-1.17C. sp. 3 frondescens 1.29-1.53 8.45-9.53 2.98-3.43C. sp. 4 frondescens 0.71-0.94 5.9-6.66 1.55-1.63C. sp. 5 frondescens 0.24-0.59 5.9-6.05 0.81-0.81  51 "Corallina sp. 1 frondescens" as "Corallina chilensis" from this point forward, and I use sequences from voucher specimen PTM 332 to represent C. chilensis in subsequent analyses.  While the C. chilensis holotype was only included in one of the rbcL trees (Figs. 8-9), all other gene trees showed a clade of closely related sequences centered around C. chilensis specimen PTM 332 (Figs. 10-22). Nine C. chilensis specimens formed a clade with 94.5/73/1 branch support in the psbA phylogeny (Figs. 10-11). Three C. chilensis specimens formed a strongly supported clade in the CO1 phylogeny with 99.4/96/1 branch support (Figs. 12-13). In the rbcL phylogeny that did not include short 1800’s herbarium sequences, the two C. chilensis specimens formed a strongly supported clade with 96.2/99/1 branch support (Figs. 14-15). The C. chilensis clade was likewise supported by the concatenated gene tree (Figs. 20-22), with 87% bootstrap support, and the majority rule tree in which C. chilensis sequences formed a clade (Figs. 16-19).   1.3.3 Other specimens thought to have been C. chilensis based on morphology  The rbcL sequence from the 1800’s d’Orbigny herbarium material matched the sequence from the C. chilensis holotype (100% identical over 263 bp) (Figs. 8-9). However, the rbcL sequence from the 1800’s Gay specimen (Fig. 5, collection unknown) differed by 0.76% (2 bp different over 263 bp) from the C. chilensis Darwin sequence (#2151). The sequence from the Gay specimen was identical to a sequence from a specimen (PTM870) called C. sp. 2 chile over 263 bp and was situated within the clade including C. ferreyrae and C. sp. 1 chile, with 99/74/.94 branch support (Figs. 8-9).    The two 2019 samples collected in Chile and identified in the field as C. officinalis var. chilensis did not match C. chilensis sequences when compared side by side, or via nucleotide   52 BLAST in GenBank. Based on psbA PTM 1985 (Appendix II, Tables S1 & S3) was only 0.06-0.18% (1-2 bp over 851 bp) different from C. sp. 2 chile, but was 1.23-1.35% (11-12 bp over 851 bp) different from C. chilensis. Also based on psbA, PTM 1984 (Appendix II, Table S1) appeared to be a species in another genus that has yet to be described. Specimen PTM 1984 was 8.83% different (71 bp different over 796 bp) from C. chilensis in the psbA gene.  The psbA sequence contributed by Paul Gabrielson from his Playa Cocholgue, Chile collection (NCU 656905) was identified as C. chilensis because it was 99.88% similar with PTM 332 across 851 basepairs (Appendix II, Table S1).  1.3.4 Analysis of conflict and congruence among gene trees   In the concatenated gene tree, which included all sequenced vouchers from Appendix II and Table S1 (except for PTM 826 (UBC A91600)) and the herbarium specimens from the 1800’s), species-level groups were monophyletic, often with strong support (Figs. 20-22). In the majority rule of the bootstrap consensus trees from the three individual loci that likewise includes all sequenced vouchers (except for PTM 826 and 1800’s herbarium specimen sequences) (Figs. 16-19), most sequences still clustered together by species name, indicating good resolution at the species level. However, relationships among Corallina species were unresolved, appearing as a polytomy of 19 clades (Figs. 16-19).  Inconsistency in reconstructing relationships by different loci was evident at various levels in the trees. Deep in the phylogeny, branching order among species varied across gene trees (Figs. 10-15). In the psbA tree, Corallina sp. 3 frondescens was sister to the clade containing the remainder of the Corallina genus (Figs. 10-11). In the CO1 tree, the C. crassissima complex clade was sister to the clade containing the remainder of the genus (Figs.   53 12-13). In the rbcL tree, the C. ferreyrae complex clade was sister to the remainder of the genus (Figs. 14-15).  Inconsistent topology among the terminal nodes was seen in the inconsistent topological arrangement of taxa within the C. ferreyrae clade across loci (Figs. 10-15). In the psbA tree, C. ferreyrae-like was sister to a clade containing C. ferreyrae sister to C. sp. 2 chile (Figs. 10-11). In the CO1 tree (Figs. 12-13), C. ferreyrae-like was sister to C. ferreyra. In the rbcL tree (Figs. 14-15), a polytomy was formed by C. ferreyrae, C. ferreyrae-like, and a clade containing C. sp. 2 chile and C. sp. 1 chile (Figs. 14-15).    I further explored the lack of resolution with respect to relationships for C. officinalis. Corallina officinalis had a different sister in each of the three gene trees (Figs. 10-15) as already mentioned in section 1.3.1. Corallina officinalis appeared as sister to the C. ferreyrae complex with 77.3/5/.64 (aLRT percent value/bootstrap percent value/Bayesian posterior probability) branch support in the psbA gene tree (Figs. 10-11). However, C. officinalis clustered sister to C. vancouveriensis with 97.5/98/1 branch support in the CO1 gene tree (Figs. 12-13). In the rbcL tree, C. officinalis clustered sister to the remainder of the genus with the exception of the C. sp. 3 frondescens complex with 88/66/1 branch support (Figs. 14-15). These are a couple of examples, but incongruence was widespread across individual gene trees (Figs. 10-15).  This incongruence was especially evident in the failure of C. officinalis sequences to form a single monophyletic group when sequences were not concatenated (Figs. 20-22). Instead, C. officinalis sequences formed three different clades by gene in the concatenated tree (Figs. 20-22). I tested for congruence of the psbA, CO1 and rbcL genes of C. officinalis by aligning concatenated genes from all taxa except C. officinalis. I added the psbA, CO1 and rbcL genes of   54 C. officinalis as separate OTUs (Operational Taxonomic Units) rather than concatenating them. If the gene genealogies were congruent, I predicted that the individual genes from C. officinalis would, if resolved, form a monophyletic or paraphyletic group in the concatenated gene tree. Instead, C. officinalis psbA, CO1 and rbcL genes each formed a sister relationship with a different clade (Fig. 20-22). I defined conflict as incongruent branches with more than .6 posterior probability and more than 60% bootstrap or aLRT support. Since C. officinalis had a different sister species relationship in each gene tree with fair to strong branch support for all three C. officinalis/sister combinations, the three gene trees were clearly in conflict (Figs. 10-15).   Beyond individual examples, overall incongruence was remarkably widespread throughout the genus, as demonstrated by the collapsed branches in the majority rule consensus of individual gene bootstrap trees (Figs. 16-19). The percentages on the branches in the majority rule consensus tree indicated the frequency that the particular topology appeared across all three individual majority rule gene trees (e.g. 33% indicated that a branch only appeared in one of the three majority rule gene trees). Some of the low support values resulted from missing data in one or more genes (Figs. 16-19). However, much of the low support resulted from disagreement across the genes. There was only one instance where all three gene trees agreed and that was with respect to how three of the outgroups clustered (Fig. 16).    Short branches mostly near the bottom of the majority rule tree (Figs 16 & 19) or otherwise nonsensically paired with other taxa were an artefact of missing data (Figs. 16-19). I confirmed this by aligning questionable sequence pairs and counting basepair differences. For example, the CO1 sequence of C. vancouveriensis JQ6158 clustered with the outgroup Crusticorallina muricata (Figs16 & 18) partly because it lacked data in the psbA and rbcL gene trees. Thus, the positions of short branches were (and should be) generally disregarded.    55 1.3.5 Distribution of C. chilensis   It is evident from the recent confirmed range of C. chilensis based on DNA sequences that the species has a fairly continuous distribution in the Northeast Pacific and has been found in two localities in Chile. Corallina chilensis has never been reported from tropical waters in the East Pacific and appears to be absent from this region. The southernmost point of its confirmed range was Playa Cocholgue, Concepción, Chile; and the northernmost point of its confirmed range was Haida Gwaii, British Columbia, Canada (Figure 23.) All C. chilensis specimens included in this analysis were verified either via DNA comparison in phylogenetic trees or in GenBank (See Appendix II, Tables S1 & S3). The C. chilensis specimens included in this analysis from the Martone collection (Appendix II, Table S1) were collected in California and between Yaquina Head, Oregon, United States; and Calvert Island, British Columbia, Canada (Fig. 23). Samples were collected specifically from the Hakai Conservancy on Calvert Island (N = 15), near the Bamfield Marine Science Centre on Vancouver Island (N = 4), in Port Renfrew located on the southern outer coast of Vancouver Island (N = 2), and from Yaquina Head, Oregon (N = 1). Hind & Saunders (2013A) likewise collected C. chilensis (as C. frondescens) from British Columbia (N=87) and northern California (N=4). Although specifically sought between southern California and Valparaiso, Chile, only one known specimen of C. chilensis has been found or documented to date by the Martone Laboratory or collaborators (Hind & Saunders 2013A; Gabrielson, pers. comm.). This specimen was collected in the drift in 2008 by Paul Gabrielson, about 700 KM south of Valparaiso, Chile, in Playa Cocholgue, Concepción, Chile (NCU656905, Appendix II, Table S3).    56  Figure 23. Recently confirmed range of C. chilensis. https://viewer.nationalmap.gov/advanced-viewer/       C. chilensis Confirmed distributionVancouver, CanadaValparaiso, ChileMonterey, CaliforniaHaida Gwaii, CanadaJuneau, AlaskaPlaya Cocholgue  57 1.3.6 Morphological measurements     The morphology of C. chilensis populations in the Northeast Pacific was highly variable (Table 4), and DNA was used to confirm specimen identities (Appendix II, Table S3). Morphometric measurement analysis revealed that the tallest fronds in each sample (Fig. 7A) were slightly longer and wider than the randomly selected fronds (Fig. 7B) that were measured, but that the width to length proportions were nearly the same (Table 4). For the tallest frond per sample, maximum height was 16-116 mm (average = 51 mm, Table 4). For the randomly selected fronds, height was 14-95 mm (average = 41mm, Table 3). Frond crowns (Fig. 7C) were 11-87 mm long (average = 31mm, Table 3), and stems (Fig. 7C) were 0-28 mm long (average = 10 mm, Table 4). Secondary pinnate branches growing from the main axis (Fig. 7D) were 6-38 mm long (average = 15mm, Table 4). Nearly half of the measured samples had secondary branches that were 5-10 mm long with three outliers 30-40 mm long. Average values for secondary branch mid-intergenicular dimensions (Fig. 7F) were minimum 0.7 mm wide, maximum 1.4 mm wide, and 1.6 mm long (Table 4). Average values for mid-intergenicular dimensions on the main axis (Fig. 7E) were minimum 1 mm wide, maximum 1.6 mm wide, and 1.6 mm long (Table 4). The length of mid-intergenicula on the secondary branches were on average less than 0.1 mm different from the length of the intergenicula on the main axis (Fig. 7E-F, Table 4). Basal intergenicula on the main axis (Fig. 7G) averaged 1.2 mm wide with a median length of 1.3 mm (Table 4). Conceptacles (Fig. 7H) averaged 0.6 mm at their widest point, and were 1.5 mm long including subtending intergenicula (Table 4). A complete list of measurements may be found in Appendix III.     58 Table 4. Summary table of morphological measurements from Corallina chilensis specimens  collected in the Northeast Pacific (N=22).   1.3.7 Morphological description of C. chilensis in the Northeast Pacific    Kützing described Corallina officinalis [var.] chilensis as tripinnate, having oblong wedged joints, “sterile” [non-reproductive] pinnules on both sides [of the branch], pointed cystocarps [conceptacles], red-violet color, [from] Chile (Kützing 1858, see Fig. 6 for original Latin text). This description likewise applied to C. chilensis collected from the Northeast Pacific. I have designated PTM 789 (UBC A91532) and PTM 333 (UBC A89285) from Calvert Island, British Columbia (Appendix II, Table S1) to serve as exemplar specimens representing Northern Hemisphere populations. I selected these two specimens because visually they were representative of all the specimens I examined; PTM 333 being symmetrical and orderly looking, while PTM 789 was asymmetrical and erratic in appearance.   Corallina chilensis fronds in the Northeast Pacific were typically 4 to 5 cm tall, but exhibited growth up to 12 cm. Crowns were nearly twice as long as stems regardless of frond height, lending some fronds a unique, feathered shape (Fig. 24 A, B & D; Fig. 27). These pinnate Average (mm) Range (mm)Frond width, random 23.93 8.97 - 51.17Frond length, random 41.32 14.24 - 95.2Frond width, tallest 29.09 7.19 - 60.1Frond length, tallest 50.59 15.98 - 115.31Crown length 31.47 11.08 - 87.2Stem length 9.85 0 - 28.39Main axis mid intergeniculum, maximum width 1.62 1.08 - 2.05 Main axis mid intergeniulum, minimum width 0.97 0.62 - 1.44Main axis mid intergeniculum, length 1.56 1.23 - 2.41Basal intergeniculum, width 1.25 0.69 - 1.83Basal intergeniculum, length 1.28 0.69 - 2.32Secondary branch length 14.91 6.49 - 37.92Secondary branch, mid intergeniculum, maximum width 1.38 0.89 - 2.49Secondary branch, mid intergeniculum, minimum width 0.68 0.41 - 1.07Secondary branch, mid intergeniculum, length 1.64 1.17 - 2.02Conceptacle width 0.65 0.61 - 0.71Conceptacle length 1.52 0.96 - 2.54  59 feather-like fronds had unbranched “stems,” and exhibited regular opposite branching about 1/3 of the way up the length of the frond. Fronds typically exhibited pinnate or bipinnate branching patterns, but some exhibited tripinnate branching.   Branching was always distichous in C. chilensis. In some individuals the main axis was dichotomously divided early in development near the base. Any clumping appearance was due to layers of branching and multiple degrees of branching, but the branches were distichous. Individuals looked spindly or robust depending on branch thickness and gap size between branches (Fig. 24). Some had secondary branches that were so broad, little to no space was visible between the branches (Fig. 24, B & D). Other specimens had narrower branches, thus larger gaps between branches, giving fronds a sparse appearance (Fig. 24, A, E, & F). Many specimens appeared to be symmetrical and orderly looking (Fig. 24, A, B, & D). Incongruent development of secondary or tertiary branching and damaged branches may have contributed to giving other specimens an erratic, irregular look (Fig. 24, C, E-F). Terminal peripheral intergenicula ranged in form from thin and rod-shaped to broad and nearly palmate.    60  Figure 24. Corallina chilensis from Martone collections from British Columbia, Canada. Scale bars represent 1 cm. Specimens are morphologically variable despite growing in the same region. (A) PTM 789 (UBC A91532) Calvert Island, Fifth Beach. Mid intertidal, in tidepool. (B) PTM 487 (UBC A89808) Calvert Island, Fifth Beach channel, subtidal. (C) PTM 182 (UBC A88708) Botanical Beach, Port Renfrew, Vancouver Island. Very exposed, mid intertidal tidepool. (D) PTM 333 (UBC A89285) Calvert Island, Fifth beach, exposed point. Low intertidal. (E) PTM 629 (UBC A89961) Botany Bay, Port Renfrew, Vancouver Island. Mid intertidal tidepool. (F) PTM 326 (UBC A89279) Calvert Island, Fifth beach. Low intertidal tidepool.   Figure 25 shows Corallina vancouveriensis fronds side-by-side with C. chilensis fronds, including at higher magnifications. While it is difficult to generalize and there are exceptions to every description, the C. chilensis specimens that I observed had mid-intergenicula on secondary branches off the main axes that tended to taper downwards distinctively. These midaxis-intergenicula possessed clear minimum and maximum width points characteristic of Corallina intergenicula, including C. vancouveriensis intergenicula (Fig. 25, E & F). Corallina chilensis basal intergenicula on the main axis appeared to be symmetrically square in surface view and were about as long as they were wide, similar to C. vancouveriensis basal intergenicular shape (Fig. 25, A & B). However, with the exception terminal peripheral intergenicula and basal intergenicula, all C. chilensis intergenicula tended to be well over 1 mm in length, 1mm at their widest points, and over .5 mm at their narrowest points (Fig. 26). A B CED F  61  Figure 25. Corallina vancouveriensis and C. chilensis side-by-side comparison. (A) Macroscopic C. vancouveriensis, scale bar ~10mm, PTM 179 (UBC A88705). (B) Corallina chilensis, scale bar ~10mm, PTM 333 (A89285). (C) Corallina vancouveriensis, scale bar = 2mm. (D) Corallina chilensis, scale bar = 2mm. (E) Corallina vancouveriensis, scale bar = 200µm. (F) Corallina chilensis, scale bar = 200 µm.    10 mm black lines A&B2 mm C&D200 um E&FCDE FC. vancouveriensis C. chilensisACPTM 179 C. vanB  62  Corallina chilensis had noticeably larger mid-main axis and mid-secondary branch intergenicula than C. vancouveriensis with respect to all three dimensions—length, maximum width, and minimum width—of intergenicula that were neither apical or basal (Fig. 26). This difference is so striking that it is even apparent in side-by-side photographs of the two species (Fig. 25). With respect to habitat, C. chilensis was often found in the low intertidal zone under Phyllospadix spp. or kelp whereas C. vancouveriensis was found either under kelp in the low intertidal or growing exposed on rocks and around rims of mid-intertidal pools.      Figure 26. Corallina chilensis (N=22) and C. vancouveriensis (N=19) intergenicular dimensions. All measurements taken from intergenicula located midway up the main axis.    Intergenicula length (µm)Intergenicula max width (µm)C. chilensis C. vancouveriensis C. chilensisC. chilensis C. vancouveriensisC. vancouveriensisIntergenicula min width (µm)C. vancouveriensisC. chilensis C. chilensis C. vancouveriensisP < 0.001P = 0.685Frond length (mm)Conceptacle width (µm)A BC D EP < 0.001 P < 0.001 P < 0.001D  63  In summary, the C. chilensis populations measured from the Northeast Pacific (Appendix III, Table S4) are unified by their overall frond shape, distichous branching, and typically, opposite pinnate branching (Figs. 24, 25B & D) and are distinguished from C. vancouveriensis by their larger mid-axis intergenicula (Fig 25). However, specimens even within the same area may appear very different from each other due to differences in overall symmetry, frond width and height, branch thickness, differing degrees of pinnateness, and variability in shape of the small peripheral branches.    Figure 27. In situs photograph in which C. chilensis fronds were growing in an exposed location. North Beach, Calvert Island, British Columbia, Canada. July 26, 2017.  PTM1588 (UBC A93226)(Appendix II, Table S1).           64 1.4 Discussion   1.4.1 Identity and rank of C. chilensis   DNA sequencing has been increasingly used over the past decade to discriminate and identify species of articulated corallines, particularly as it has become more evident that exclusively morphologically-based species are inadequate due to the presence of cryptic species, as well as morphologically variable speciation. This could be on account of convergent evolution or phenotypic plasticity. Even when morphological boundaries are known to broadly align with molecular-based species boundaries, analyzing DNA sequences increases the resolution into the relatedness of taxa. Because random mutations in the genome are not always reflected in the phenotype, by directly comparing base pair differences in the DNA itself, we gain an increased level of resolution to a degree that morpho-anatomic analyses simply are not capable of achieving. While DNA sequences may be compared along-side one another base by base to determine the percent difference between specimens or species, DNA sequences also may be incorporated into phylogenetic analyses used to detect reciprocal monophyly, a criterion confirming that any given population is a distinct “species.” Distance matrices may be used to compare DNA sequences and may indicate divergence of intermediates even if newly emerging species do not yet demonstrate reciprocal monophyly in phylogenetic analyses or exhibit distinguishing morphological features. DNA sequences may also be analyzed using species discrimination programs such as Automatic Barcode Gap Discovery. Such programs delimit species based on greater variation between (interspecific) than within (intraspecific) any given set of diverging populations or species. Inclusion of DNA from multiple loci has the potential to strengthen evidence for speciation, given that no universal barcode has been demonstrated to effectively segregate coralline species (Broom 2008, Leliaert 2014). For this reason, I selected   65 three different markers from two different organelles (psbA-plastid, CO1-mitochondrial, and rbcL-plastid) that have been commonly used in previous analyses, as well as for the sake of comparison between my analysis and analyses completed by other researchers. Once species boundaries are determined, it is necessary to compare DNA from field-collected specimens with the DNA from type specimens in order to correctly apply species names. To this end, I included type specimen DNA from Corallina chilensis in my analysis to ensure correct name application.   This study established that a partial rbcL sequence from the holotype specimen of C. chilensis, basonym of C. officinalis var. chilensis, from Valparaiso, Chile was identical over its length with PTM 332 (UBC A89284, Appendix II, Table S1) from Hakai, British Columbia, Canada. PTM 332 in turn matched ~100 other specimens included in this study that were collected from the Northern Hemisphere (Fig. 23, Appendix II, Tables S1 & S3), more specifically from the Northeast Pacific from the Haida Gwaii archipelago in British Columbia through Laguna Beach, California, USA, and one other specimen (NCU656905, Appendix II, Table S3) collected from the Southern Hemisphere, from Playa Cocholgue, Concepción, Chile.  Overall, evidence that C. chilensis is a “separately evolving metapopulation” (De Queiroz 2007) and thus a distinctive species includes: (1) C. chilensis specimens form well-supported clades based on phylogenetic analyses of each of the three markers; (2) C. chilensis is molecularly distinct from other species within its genus based on sequence divergence values for each of the same markers (Table 3, and see Appendix V); (3) C. chilensis was shown to be an independent species from C. officinalis using CO1 and rbcL gene sequences in ABGD analyses (4) C. chilensis exhibits a biogeographic range and phenotype distinct from C. officinalis (Brodie et al. 2013, Hind et al. 2014A). (5) Northeast Pacific C. chilensis specimens examined were morphologically different from other species including congeneric species C. vancouveriensis.    66  Given the DNA-confirmed result that C. chilensis has been collected from several locations in the Northeast Pacific including in the same locale from which Yendo collected, and that it has been collected twice from Chile; Yendo (1902) was correct: C. chilensis is present in both hemispheres. Further, based on the evidence provided above, C. chilensis is a distinct species and cannot be considered a variety of C. officinalis as proposed by Kützing (1858) and accepted by numerous, although not all, subsequent researchers. While the main questions introduced in this thesis have been answered, these answers raise new questions that merit discussion.  1.4.2 Known global distribution of Corallina, and specifically C. chilensis   Members of the subfamily Corallinoideae are distributed world-wide (Guiry & Guiry 2020) and the Corallina genus as a whole exhibits an extensive global distribution (Broom et al. 2008, Walker et al. 2009, Brodie et al. 2013, Williamson 2015, Bustamante 2019, Guiry & Guiry 2020). Other genera in subfamily Corallinoideae are also widespread. For instance, Bossiella species are found in both the Northeast and Southeast temperate Pacific, but only one Bossiella species is known to span both hemispheres (Hind et al. 2014B, 2015, 2018). Jania has been confirmed (with DNA sequences) to be present in both the Atlantic and Pacific oceans, in South African waters, and surrounding Australia (Kim et al. 2007, Harvey et al. 2020).  Even individual species within Corallina are widespread. Corallina officinalis has been confirmed in the North Pacific and the North Atlantic including as far North as Iceland south to Spain (Yesson et al. 2018), also extending into the Southern Hemisphere (Broom et al. 2008). Corallina ferreyrae grows in the Northeast Pacific and in Chile, and may possibly inhabit the North Atlantic as well, depending on the criteria defining species (Appendix II, table S1; Walker   67 et al. 2009, Bustamante et al. 2019). Like these congeners, C. chilensis also exhibits an extensive range.   Corallina chilensis, identified only using morphology, was reported specifically in Valparaiso, Chile (Harvey 1849), Puerto del Hambre (Port Famine) in the Strait of Magellan (Montagne 1852), Magellanes province, Chile (Ardissone 1888), Bahia Orange, Chile (Hariot 1889), Tierra del Fuego, Chile (Foslie 1907), Beagle Channel, Southern Chile north through Lima (Ramírez & Santelices 1991), Berkeley Sound, Port Louis, Falklands (Foslie 1907), Norfolk Island, Australia (Harvey 1849), Port Renfrew, British Columbia, Canada (Yendo 1902A), South Africa (Silva et al. 1996), Hakodate, Japan (Yendo 1902B); and reported as “common” on the coast of California (Setchell & Gardner 1903), “common” from San Diego, California north to Vancouver Island, Canada (Smith 1944), “common” all along the coast of Mexico from Isla Magdalena, Baja Mexico Sur, north to British Columbia, Canada (Dawson 1953). Collectively these reports depict a nearly continuous range from the Falklands and southern Chile through Vancouver Island, Canada, with the exception of presence in the tropics. My study used DNA sequences to confirm the presence of C. chilensis in some, but not all, of these locations because not all historically sampled sites were resampled, or C. chilensis was not present at the sites that were sampled recently, in the past few decades. This study confirmed the presence of C. chilensis in the Northeast Pacific as far south as Laguna Beach, California. A specimen was also collected from Yaquina Head, Oregon, but the majority of specimens were collected from waters surrounding Vancouver and Calvert Islands, British Columbia, Canada. Sequences taken from Hind and Saunders (2013A) were from specimens collected from Haida Gwaii, Canada, and matched the C. chilensis sequences from my study. This indicates that C. chilensis is distributed at least as far North in the Northeast Pacific as Haida Gwaii, British   68 Columbia, Canada. Additional sampling is required to determine the northern most boundary for C. chilensis. With respect to Southern Hemisphere distribution, sequencing the Darwin material confirmed that C. chilensis was present in Valparaiso, Chile, in the early 1800’s, and this research also confirmed that it is still present in Chile, ~700 km South of Valparaiso in Playa Cocholgue, Concepción.   Early C. chilensis species identifications were made based exclusively on morphology, and without direct comparison to the type specimen. Even if these collections had been morphologically compared with the type specimen, that still would not have guaranteed correct name application, as was the case with the Gay specimen which closely resembled the C. chilensis Darwin specimen, but was not conspecific when confirmed using DNA. While in some geographic locations it may be sometimes possible to discern C. chilensis from other neighboring corallines based on morphology, it may not be possible to do so in other geographical locations where different species are present and or cryptic with one another. Thus, without DNA confirmation, there is no way of verifying the identifications in the historical reports based only on morphological identifications, and therefore the historical range of C. chilensis is poorly documented.   Even if historical reports of C. chilensis presence between California and Valparaiso were inaccurate, this range is consistent with other algae that exhibit similar disjunct ranges. The articulated coralline Bossiella orbigniana ranges from Haida Gwaii Canada, south to Baja California Norte, Mexico (Hind et al. 2014B). Callophyllis variegata has likewise been confirmed (by DNA), and ranges from Monterey, California through Haida Gwaii, British Columbia, although its type specimen was from Valparaiso, Chile, and it was collected recently from Ancud Bay and Los Chonos Chiloé, Chile (Clarkston & Saunders 2013). Similarly,   69 Mastocarpus latissimus was confirmed in Chile, but otherwise ranges from Moss Landing, Monterey Co., California, north through Alaska (Lindstrom et al. 2011).   If more C. chilensis specimens were collected from the Southern Hemisphere, and if quality DNA could be extracted from old herbarium specimens, it would then be possible to conduct genetic studies on both Southeast and Northeast Pacific populations to understand the extent of their genetic separation, if they did indeed display any dissimilarity. If haplotypes were the same between the two populations, the indication would be that the two populations had only recently diverged, and that one was thus more recently (perhaps in the past few centuries) introduced to the opposite hemisphere. Alternatively, different haplotypes between the two populations would indicate that separation occurred over much deeper time. Knowing how recently the populations were separated could potentially provide clues as to how C. chilensis was distributed across its range. For instance, if the introduction of C. chilensis to the opposite hemisphere had been recent, in the past few centuries, maritime traffic could have been responsible for its introduction to locations far from its origin (Callahan et al. 2001, Ruiz et al. 2003, Mach et al. 2017, Goldsmit et al. 2018), anomalous cooling events might have impacted its distribution in the Southern Hemisphere (Thompson et al. 1986, 2003, Meyer 2009), or perhaps a rare event in which C. chilensis fronds could have become entangled in and transported via kelp raft might have occurred (Saunders 2014).    1.4.3 How to identify C. chilensis in British Columbia, Canada   I recommend first attempting to identify the genus of a given unknown geniculate coralline that could potentially be C. chilensis. Reproductive C. chilensis exhibits the typical Corallina shaped bulbous axial conceptacles, and non-reproductive specimens may still be   70 placed into Corallina based on the typical shape of mid intergenicula on the main secondary axes (see Abbott & Hollenberg 1976, Johansen 1981, Baba et al. 1988) distinguishing them from certain other genera, such as Bossiella, Calliarthron, Chiharaea, and Johansenia. (For a description of these other genera containing species that fall within the same range as C. chilensis from British Columbia, see Hind et al. 2014A and Hind et al. 2015 for descriptions of Bossiella species; Gabrielson et al. 2011 for Calliarthron species, Martone et al. 2012 and Hind & Saunders 2013B for Chiharaea species; Hind & Saunders 2013A for Johansenia.) This typical Corallina shape occurs because width is shorter than length and the intergenicula taper downwards decreasing in width. Variation in branching characteristics likely contributes to the overall variable appearance of the C. chilensis population from the same region. However, the consistent shape of the central intergenicula is a unifying feature across otherwise enigmatic morphological variation.    Many described Corallina species do not occur in the same geographic range as British Columbian C. chilensis, or are not likely to be mistaken for C. chilensis on account of distinct morphological differences (Walker et al. 2009, Martone et al. 2012, Hind & Saunders 2013A, Hind et al. 2014A, Bustamante 2019). The only other described and DNA-confirmed members of Corallina present in British Columbia growing in the same geographical range as C. chilensis are C. vancouveriensis and C. officinalis. Corallina officinalis only occurs in its ‘Pachyarthron’ morphology within that range (Hind et al. 2014A) making it highly unlikely that it would be mistaken for C. chilensis because it looks similar to Calliarthron. This leaves mostly only C. vancouveriensis and C. chilensis to be mistaken for one another. There are some other provisionally named species that resemble and could possibly be confused with C. vancouveriensis, and this analysis did not compare species from other genera that could   71 potentially be mistaken for Corallina from time to time. While C. chilensis exhibits similar intergenicular shape to other Corallina species, some specimens may be reliably differentiated in the field without a microscope from neighboring C. vancouveriensis based on the immense intergenicula size difference. While frond length is an insignificant diagnostic characteristic, the best features to look for when differentiating between the two species is intergenicular length, maximum intergenicular width, minimum intergenicular width, and conceptacle width. Conceptacles branching from C. chilensis may appear small for Corallina conceptacles, but only because C. chilensis genicula are so large, creating the illusion that the conceptacles are smaller. On average, C. chilensis conceptacles are actually ~0.1 mm wider than C. vancouveriensis conceptacles.  1.4.4 Phylogenetic position of C. chilensis within Corallina     While all three gene trees support C. chilensis as a distinct species, they do not resolve the relationship of C. chilensis with other species in the genus. Corallina chilensis is sister to different Corallina species depending on the gene analyzed. Many of these ambiguities will be illustrated in detail in the Future Directions chapter of this thesis. While C. chilensis is clearly not the most closely related species (sister) to C. officinalis, the relationships between C. chilensis and other species in the genus are not well supported or consistent across trees.   In spite of their polytomies, the concatenated and majority rule trees show consistent groupings of sequences into species groups based on the inclusion of more data in the trees. In the concatenated analysis, C. officinalis sequences were left as separate operational taxonomic units rather than concatenating them, for the purpose of evaluating whether different loci sequenced from the same specimens occurred in the same clade with C. chilensis. Indicating   72 disagreement across the three individual gene trees, C. officinalis sequences from each locus had a different sister relationship in the concatenated tree. None of the three single-locus clades of C. officinalis formed a sister relationship with C. chilensis, indicating that C. chilensis cannot be considered a variety of C. officinalis.  Corallina chilensis was delimited as a distinct species in both the CO1 and rbcL ABGD analyses, but there was lack of evidence for separation in the psbA ABGD analysis. The context of this result should be taken into consideration. While psbA is used because it is easy to amplify, it is the least variable of the three loci that I used and it offered the least resolution of relationships at the species level. Additional alternative markers are required to provide better resolution for the purpose of species delimitation (Broom et al. 2008). Thus, the lack of evidence in the ABGD psbA analysis for delimitation of C. chilensis as a species separate from a group of four others including C. officinalis is probably a function of psbA’s low information content (Zhan et al. 2020).  Pairwise percent differences further support the distinction of C. chilensis from other described or provisional (undescribed) species, exceeding thresholds listed in previous publications of Corallina and other genera in Corallinoideae (Martone et al. 2012, Hind & Saunders 2013A, Hind & Saunders 2013B, Hind et al. 2018). Interestingly, despite C. sp. 5 frondescens being highly similar to C. chilensis with respect to psbA and rbcL sequences, the two species exceeded previously described percent difference thresholds for species delimitation with respect to CO1 (Hind & Saunders 2013B, Hind et al. 2018).       73 1.4.5 Incongruence across coralline gene trees: an anomaly or more common than we think?    The relationships among species in individual gene trees were rife with topological incongruence. The purpose of the majority rule tree was to illustrate the great amount of conflict across individual trees and the breakdown of deeper structure. While C. chilensis formed a clade in the majority rule tree, C. officinalis sequences still consistently clustered in primarily three different locations  although this result was confounded by the low support values and the presence of zero-length branches. However, the concatenated tree, consistent with the majority rule consensus tree shows C. officinalis sequences segregated by gene into different clades. Corallina officinalis provides one example of the widespread incongruence across the three Corallina gene trees. The abundance of polytomies throughout all phylogenetic analyses and disagreement indicated by collapse of backbone structure in the majority rule tree testify to the conflict among gene trees for almost every species within Corallina.  As in my study, incongruence has been noted in previous studies. Contradictory topologies are evident in previously published trees for Corallina (see Figs 1-3 in Hind & Saunders 2013A). However, it is difficult to determine how widespread such disagreement is within genera across Corallinoideae. Several genera have few known species, e.g., Ellisolandia (Hind & Saunders 2013A) and Johansenia (Hind & Saunders 2013A) each have only one species, and Calliarthron and Alatocladia each have only two species (Gabrielson et al. 2011). In the case of Crusticorallina with four species, the SSU gene tree and the concatenated psbA-CO1-rbcL gene tree have different topologies, but this may be attributable to the SSU gene’s lack of resolving power. Low bootstrap support in the SSU tree suggests that some of the conflicting branching order may reflect stochastic variation rather than strong phylogenetic signal. Differences in taxon sampling, especially of outgroups may also have contributed to   74 conflict (Hind et al. 2016, see Figs 1-2). Upon examination of the individual gene trees, psbA and rbcL trees were in agreement, but the CO1 tree disagreed with the psbA and rbcL trees (Hind et al. 2016, see supplementary materials).  There is some evidence of incongruence within Bossiella, a genus with ~14-17 species, comparable in size to Corallina. However, individual trees from each marker are not always presented in publications on Bossiella, and even in the same publication, tree topologies are sometimes difficult to compare because they were generated using different methods (Hind et al. 2014, Hind et al. 2015, Hind et al. 2018). While the differences between the trees could be attributable to the different analyses and outgroups, a large polytomy and mediocre branch support in the concatenated tree in Hind et al. (2014) could also be related to disagreement between psbA and CO1 (see Figs. 1-2 in Hind et al. 2014). For future endeavors, I would recommend including rigorous analysis of individual genes and presentation of individual gene trees in publications, or including them in supplemental materials sections so that the degree of conflict between gene trees may be better ascertained.   Assuming that each plastid and mitochondrial genome consists of a single chromosome that is uniparentally inherited, I would expect individual plastid or mitochondrial gene genealogies to be congruent (Janouškovec et al. 2013, Muñoz-Gómez et al. 2017, Lee et al. 2018, Yoshida & Mogi 2019). So it was surprising to discover such strong and widespread discordance between two plastid genes (psbA and rbcL) presumably located on the same chromosome. Incongruence has recently been more thoroughly documented among non-algal taxa (Moncalvo et al. 2006, Bell & Hyvönen 2009, Cranston et al. 2009, Moyer et al. 2009, Pelser et al. 2010, Jarvis et al. 2014) but there are few explicit references to it with respect to individual gene tree topologies in the red algal literature (Lee et al. 2018, Zhan et al. 2020). For   75 Rhodophyta, this is likely due to limited genomic sampling (Janouškovec et al. 2013, Lee et al. 2016, Muñoz-Gómez et al. 2017). The findings from the handful of studies that have extensively examined red algal genomes cited high genomic diversity, evidence of horizontal gene transfer, transposons (Janouškovec et al. 2013, Muñoz-Gómez et al. 2017), and one study cited evidence that ancient red algal plasmids spread as parasitic genetic elements, a.k.a. “selfish genes” (Lee et al. 2016). Given that such means of potential genomic flexibility have been detected among the few taxa that have been studied, undetected conflict may be more prevalent than currently thought. An apparent lack of conflict could reflect unrecognized conflict in previous studies.   Incongruence indicates differences in evolutionary histories among gene trees due to one or more phenomena. Some studies have attempted to assess incongruencies and determine their causes (Pelser et al. 2010), but approach and methodology are still under debate (Mossel & Vigoda 2005, Cranston et al. 2009, Pelser et al. 2010). Possible causes include incomplete lineage sorting, especially among early-diverged lineages after a rapid radiation; movement of genes between species perhaps via hybridization, introgression, or horizontal gene transfer; or the duplication and subsequent extinction of gene copies (Maddison 1997, Liu & Pearl 2007, Cranston et al. 2009, Moyer et al. 2009, Pelser et al. 2010, Bell & Hyvönen 2010, Jarvis et al. 2014, Lee et al. 2016, Lee et al. 2018). Hybridization and incomplete lineage sorting are difficult to distinguish (Pelser et al. 2010), and future work involving many more replicates and loci across as many species as possible would be required to tease the two influences apart (Maddison & Knowles 2006, Moyer et al. 2009, Janouškovec et al. 2013, Jarvis et al. 2014).  While some studies have used concatenated analyses despite incongruence among gene trees (Hind & Saunders 2013A, Cranston et al. 2009, Jarvis et al. 2014), other researchers advise against concatenated species trees when there is conflict among individual gene trees because   76 concatenating genes in phylogenetics has the potential to obscure distinct evolutionary histories, yielding misleading results (Mossel & Vigoda 2005, Liu & Pearl 2007). Instead of concatenating, Mossel & Vigoda (2005) and Liu & Pearl (2007) recommend reconstructing phylogeny based on each individual locus when there are conflicting signals, an approach that I used in creating three separate gene trees as well as the majority rule tree where multiple genes were included in the same tree, but sequences were not concatenated.    1.4.6 Conclusions    In conclusion, the rbcL sequence of a holotype, Darwin’s Corallina chilensis specimen from the Southern Hemisphere, was an identical genetic match with a sequence from an entity provisionally called C. sp. 1 frondescens in the Northern Hemisphere. Going forward, this entity should not be referred to as C. officinalis var. chilensis but rather as C. chilensis because that is the oldest applicable name, and because C. chilensis is not a variation of C. officinalis. I have confirmed that C. chilensis is present in both hemispheres, including near the British Columbia location where Yendo once collected. Yendo was therefore likely to have been correct, back in 1902, in his report that C. chilensis (as C. officinalis var. chilensis) was present in British Columbia.  1.4.7 Future directions with respect to C. chilensis    Study of additional specimens would be required to update the description of Southern Hemisphere C. chilensis populations and to reconstruct the full extent of C. chilensis’ range. In this thesis I provide a morphological description based on the C. chilensis populations of the Northeast Pacific because only two additional collections have been found of C. chilensis from the Southern Hemisphere. Only three specimens from the Southern Hemisphere have been   77 confirmed (using DNA) as C. chilensis to date, which is too small a sample size upon which to base a description. More collections are needed from the Southern Hemisphere, particularly from the subtidal which has not yet been extensively sampled. It is especially important to update the description of the Southern Hemisphere C. chilensis populations given how the original morphological description of C. chilensis may have been based in part on the 1800’s Gay collection, which as I have shown, does not represent C. chilensis.  While I have completed a fairly comprehensive search of the literature regarding C. chilensis, more research has the potential to establish the historical range of the species and to determine if the species’ range or abundance has shifted since the early 1800’s. The historical ranges could be inferred by obtaining and mapping the reported collection localities of C. chilensis from herbarium records. Reports could be traced to specimens, where possible. To establish the identity of herbarium specimens as bona fide C. chilensis, DNA from them should be extracted and sequenced. Sites where bona fide C. chilensis was collected historically (e.g. from Southern Chile and the Falkland Islands in the 1800’s) should then be thoroughly resampled, to assess whether C. chilensis is now present or absent at those locations.               78 Chapter II Future Directions in Corallina   2.1 Introduction   Corallina chilensis is just one of many species in Corallina. A number of older Corallina species names need re-evaluation based on molecular analysis. Some of these existing names may even apply to contemporary species that currently have only provisional names.    Corallina species, including provisional species, were recognized by Hind & Saunders (2013A) primarily based on a CO1 neighbor-joining analysis with a minimum threshold of 3.3% difference in CO1 and a three-gene concatenated tree that had no strongly supported nodes within the genus (see Hind & Saunders 2013A, Figs. 1-2). The CO1 percent difference threshold of 3.3% from Hind & Saunders 2013A was updated to 4.5-5.8% in Hind et al. (2018). The lack of support in the Hind & Saunders (2013A) concatenated tree was likely a result of the incongruence across individual genes that I also detected.  Multiple lines of evidence should be used, where possible, when delimitating species. For this thesis, and to augment and improve upon Hind and Saunders (2013A), I conducted three types of analyses in an attempt to provide additional evidence for species boundaries within Corallina. I conducted phylogenetic analyses on psbA, CO1, and rbcL genes independently, as well as on multigene alignments (See Figs. 16-22 for majority rule & concatenated trees). ABGD barcoding (Table 2) and percent distance matrices (see Appendix V) were also completed as preliminary analyses, but provide supplemental support and corroborate the phylogenetic findings. Hind et al. (2018) percent difference thresholds were used as a guideline, but not as a   79 cutoff for species delimitation. Percent differences between sequences that fall below Hind et al. (2018) may indicate conspecificity or merely that the species are closely related. In this chapter, I review the results for other Corallina species besides C. chilensis.   2.2 Examination of currently accepted Corallina species   AlgaeBase (Guiry & Guiry, Retrieved May 7, 2020) lists 204 valid species names, an additional 68 infraspecific names, with 28 of these taxonomically accepted as species in Corallina. I included sequences corresponding to 9 of these species names in my analyses: C. officinalis, C. vancouveriensis, C. ferreyrae, C. aberrans, C. crassissima, C. declinata, C. maxima, C. melobesioides, and C. pinnatifolia. All three of the gene sequences were only available for some species. Some species are represented by only one or two of the genes, or by only one or a few specimens (Appendix II, Table S1).   2.2.1 Evaluation of the generitype C. officinalis Linnaeus   Sequences from the C. officinalis epitype selected by Brodie et al. (2013) were included in my study. Consistent with its delimitation as a separate species, C. officinalis sequences were monophyletic in each individual gene tree (Figs. 10-15) and they were also monophyletic when concatenated and included in a tree of concatenated genes (see Hind & Saunders 2013A, Fig. 1, concatenated tree.) The relationship of C. officinalis to other species was unresolved, due to the conflicting positions of the species in different gene trees (Figs. 10-15, 20-22).  2.2.2 Evaluation of accepted species C. vancouveriensis Yendo   Yendo originally described C. vancouveriensis in his 1902 publication on seaweeds he collected from Vancouver Island, Canada. Unfortunately his collections have not been located,   80 but a specimen in the University of California herbarium (UC) has been designated as a lectotype and has been sequenced (Unpublished data, Gabrielson, pers. comm.). I did not have access to this lectotype for my study.   My analyses support recognition of C. vancouveriensis as a distinct species. The individual psbA and CO1 gene trees demonstrated strong branch support for C. vancouveriensis on its own or in combination with C. sp. 2 vancouveriensis (Figs. 10-13). Likewise, C. vancouveriensis formed a clade in the majority rule tree (Figs. 16-19) and had fair branch support in the concatenated tree (Figs. 20-22). All three ABGD analyses delimited C. vancouveriensis as a separate species (Table 2).   2.2.3 Evaluation of accepted species C. crassissima, C. declinata, and C. aberrans (Yendo) K.R.Hind & G.W. Saunders   For some of the species that Yendo described as new to science, Yendo cited multiple localities without designating a holotype. Hind and Saunders (2013A) designated lectotypes from among Yendo’s illustrations for C. crassissima, C. declinata, and C. aberrans corresponding with basonyms Amphiroa crassissima, Amphiroa declinata, and Amphiroa abberans respectively (Yendo 1902B). The published DNA sequences that I included in my analyses were from Hind and Saunders (2013A), but are not topotype material because it is unknown which of the syntype localities corresponded with each of the designated lectotypes.  This complex as a whole was monophyletic across all gene trees (Figs. 10-15) with strong support except for in the psbA tree (Figs. 10-11). The CO1 and rbcL trees (Figs. 12-15) differentiated between closely related species within the complex, but the psbA tree (Figs. 10-11) lacked the resolving power to differentiate between species. This is likewise reflected in the distance matrices (Table 5). The monophyly of the complex indicates that it represents at least   81 one distinct species within Corallina. The taxon sampling was incomplete for C. declinata, which lacked rbcL data and limits my ability to assess its status as a species. Knowledge of morphological and ecological factors could also perhaps provide additional lines of evidence for the distinction of species within this complex.  Table 5. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.    2.2.4 Evaluation of accepted species C. ferreyrae E.Y. Dawson, O.C.Acleto, & N.Foldvik   DNA was extracted and sequenced from a C. ferreyrae isotype specimen, by Bustamante et al. (2019), from which I obtained sequences for all three of my gene analyses. My analyses support C. ferreyrae as a distinct species within Corallina. Corallina ferreyrae isotype sequences fell within monophyletic groups in all three gene trees (Figs. 10-15) with strong support except for in psbA (Figs. 10-11), in which it was only moderately supported with 44.2/58/.93 branch support. Corallina ferreyrae sequences likewise formed monophyletic groups in each of the majority rule and concatenated trees (Figs. 16-22), with strong branch support in the concatenated tree (Figs. 20-22). Discounting the outlier PTM 826 (likely contamination) from C. crassissima  psbA 0 psbA 0.7-1.3CO1 0 CO1 4.5-5.8rbcL 0 rbcL 1.6-1.9C. aberrans  psbA 0.47-0.59 psbA 0.12CO1 4.54 CO1 0rbcL 0.82 rbcL /C. declinata  psbA 0.47 psbA 0-0.12 psbA 0CO1 4.84-5.22 CO1 4.54-5.06 CO1 0.47rbcL / rbcL / rbcL /C. officinalis  psbA 0.82 psbA 0.82-0.94 psbA 0.82 psbA 0CO1 6.12-7.41 CO1 6.68-7.12 CO1 6.68-7.44 CO1 0rbcL 2.62-3.83 rbcL 3-3.98 rbcL / rbcL 0.15-0.59C. crassissima Hind et al. 2018          C. aberransC. declinataC. officinalis  82 the CO1 analysis, all three ABGD analyses (Table 2) indicated that C. ferreyrae was its own distinct species.   2.2.5 Evaluation of accepted species C. maxima (Yendo) K.R. Hind & G.W. Saunders   Corallina maxima is associated with the basionym Cheilosporum maximum (Yendo 1902B). The two C. maxima sequences included in my analyses, one psbA sequence (JQ422207) and one CO1 sequence (JQ615680) (Appendix II, Table S1), were both from the same Japanese representative specimen from Hind and Saunders (2013A). There was no DNA available from the designated lectotype specimen, as it was an illustration of a specimen from an unknown locale (Yendo 1902B). Even without rbcL data, the divergence of its psbA and CO1 sequences consistently indicated that C. maxima is genetically distinct from other species in my dataset. Corallina maxima was distinguished at the species level in both psbA and CO1 ABGD analyses (Table 2). The two sequences appeared on longer branches, particularly in the concatenated tree (Figs. 20-22), and the clade that they formed was not well supported, due to the absence of overlapping data that serves in binding the clade together. Corallina maxima was sister to C. sp. 1 gws in the psbA tree (Figs. 10-11), but sister to the remainder of the genus in the CO1 tree (Figs. 12-13) indicating incongruence as also seen among other members of the genus. More sampling of specimens, with sequencing of loci including rbcL would be advisable for delimiting C. maxima and for understanding within-species variation (Figs. 10-13, 16-22).       83 2.2.6 Evaluation of accepted species C. melobesioides (Segawa) P.T.Martone, S.C.Lindstrom, K.A.Miller, P.W.Gabrielson   Martone et al. (2012) synonymized genus Yamadaia with Corallina. In their paper, their reference herbarium specimen from which DNA was extracted was collected from near the type locale, but was not the type specimen indicated in Segawa (1955) (Guiry & Guiry 2020).  Only one rbcL sequence of Japanese C. melobesioides was included in these analyses (UBC voucher A62034, Appendix II, Table S1). The rbcL ABGD analysis (Table 2) delimited it as a species, and it appeared to be closely related to C. pinnatifolia in the rbcL tree with fair branch support (85.6/83/.98) (Figs. 14-15). Interestingly, it clustered with C. pinnatifolia among other taxa in the concatenated tree with 88% bootstrap support (Figs. 20-22), although on a longer branch than the other taxa within the cluster. This will be further discussed below in section 2.3.5. This small amount of evidence raises a possibility that C. melobesioides is synonymous with C. pinnatifolia, which could be tested with more data.   2.2.7 Evaluation of accepted species C. pinnatifolia (Manza) E.Y.Dawson    Formerly referred to as Joculator pinnatifolius (basonym), the holotype associated with C. pinnatifolia was collected by F.M. Reed in 1934 (UC #545769 UC) and has not been sequenced (Dawson 1953). The Gabrielson et al. (2011) rbcL sequence I used in my analyses was extracted from Laguna Beach, California, specimen UBC A88590 (Appendix II, Table S1).   Corallina pinnatifolia was distinct from other known species in the rbcL ABGD analysis (Table 2), clustered in isolation in the majority rule tree (Figs. 16-19), yet formed a monophyletic group with 88 bootstrap support with C. melobesiodies in the concatenated tree (Figs. 20-22) among other provisionally named species to be discussed below in section 2.3.4. Corallina pinnatifolia is closely related to, or perhaps conspecific with C. sp. 2 chile, the C. caespitosa   84 holotype, and possibly C. melobesioides (Figs. 10-22, Appendix II, Table S1). If these names were to be synonymized, C. pinnatifolia would have priority over the others given that it is the oldest name.  2.2.8 Summary of evidence supporting or rejecting currently accepted species designations   In summary, of the 9 species accepted by AlgaeBase that I included in my analyses, there were at least 6 species that could be distinguished with my data. The evidence in my analyses specifically supports C. officinalis, C. vancouveriensis, C. ferreyrae, and C. maxima as delimited species. Corallina aberrans and C. declinata are closely related, possibly conspecific, and possibly also conspecific with C. crassissima. Similarly, C. melobesioides and C. pinnatifolia are closely related and may be the same species. More sequence data from more specimens and an examination of ecological and morphological evidence is required to determine explicit boundaries within these potential complexes.   2.3 Provisionally identified Corallina species   Prior to this study, Hind and Saunders (2013A) gave provisional identifiers to seven species that have yet to be confirmed or described as new. These were: C. sp. 2 vancouveriensis, C. sp. 1 gws, C. sp. 1 california, C. sp. 2 frondescens, C. sp. 3 frondescens, C. sp. 4 frondescens, and C. sp. 5 frondescens. I further added C. sp. 1 gws-like, C. sp. 3 frondescens-like, C. ferreyrae-like, C. sp. 1 chile, and C. sp. 2 chile.   2.3.1 C. sp. 3 frondescens & C. sp. 3 frondescens-like   The C. sp. 3 frondescens complex formed a well-supported clade across all phylogenetic analyses (Figs. 10-22) with the exception of the psbA tree (Figs. 10-15), which did not have   85 strong support, possibly because of long branch attraction with outgroup Ellisolandia in the Maximum Likelihood/aLRT psbA tree.  My study supported delimiting C. sp. 3 frondescens-like as a closely related species separate from C. sp. 3 frondescens (Hind & Saunders 2013A). Corallina sp. 3 frondescens and C. sp. 3 frondescens-like sequences clustered together across all individual gene trees in the same clade with strong support. The concatenated tree (Figs. 20-22) also grouped the clade containing C. sp. 3 frondescens-like sequences as sister to the clade containing C. sp. 3 frondescens sequences with strong branch support.  Other than the ABGD psbA analysis, which did not separate the two, (Table 2), the majority of the phylogenetic and ABGD evidence strongly supported delimiting C. sp. 3 frondescens-like as a species separate from C. sp. 3 frondescens. The two were distinguishable in both CO1 and rbcL ABGD analyses (Table 2). The C. sp. 3 frondescens-like sequences clustered together exclusively in two out of three genes, a rare occurrence in the majority rule analysis (Figs. 16-19). Again, more sampling, more sequencing, and in-depth morphological analysis could provide additional support for either conspecificity or separation. Meanwhile, based on the preponderance of the evidence, I interpret C. sp. 3 frondescens and C. sp. 3 frondescens-like as two separate species that have diverged recently.  A potential 3rd species in this group was indicated by the CO1 ABGD analysis that designated a single voucher, PTM 1400, UBC A92938 as a different species from the other C. sp. 3 frondescens sequences (Table 2, Appendix II, Table S1). As this is a single sequence from one sample, more substantial evidence, from more gene sequences and from additional collections from where PTM 1400 was found would be required for confirmation that it is indeed a distinct species across other genes besides CO1. The sequence from specimen PTM 1400   86 accounts for the over two percent difference range (3.03-5.3% difference) within C. sp. 3 frondescens in the CO1 distance matrix (Table 6). Corallina sp. 3 frondescens PTM 1400/UBC A92938 likewise stood apart from all other C. sp. 3 frondescens sequences in the concatenated analysis (Figs. 20-22), but was included as a longer branch within the cluster in the majority rule tree where sequences were not concatenated (Figs. 16-19).  Table 6. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.     2.3.2 C. sp. 1 gws & C. sp. 1 gws-like    Corallina sp. 1 gws formed a sister group to C. sp. 1 gws-like with strong support across all three gene and concatenated trees (Figs. 10-15, 20-22), supporting both as a new and distinct Corallina species. Following a similar pattern, ABGD analysis of CO1 and rbcL also supported the separate species while the ABGD analysis of psbA did not resolve them as different (Table 2 & Figs. 10-11). Both taxa were distinct from C. officinalis based on percent difference across all distance matrices (Table 7) well above the comparable ranges described by Hind et al. (2018). Corallina sp. 1 gws-like was not very dissimilar from C. sp. 1 gws when comparing percent psbA 0.7-1.3CO1 4.5-5.8rbcL 1.6-1.9C. sp. 3 frondescens-like  psbA 0.41-0.59CO1 3.03-5.3rbcL 0.72-1.2C. officinalis  psbA 1.06-1.18 psbA 1.29CO1 8.32-8.42 CO1 7.42-8.02rbcL 2.62-3.24 rbcL 2.77-3.39C. vancouveriensis  psbA 1.41-1.65 psbA 1.76CO1 7.66-8.85 CO1 8.47-8.77rbcL 3.55-3.71 rbcL 3.46-3.55C. sp. 3 frondescensHind et al. 2018          C. sp. 3 frondescens-like  87 differences (Table 7), well below Hind et al. 2018 percent difference thresholds across all three genes.  The evidence described above overwhelmingly supports the species distinction of C. sp. 1 gws, one of the provisionally identified species suggested by Hind & Saunders (2013A). However, C. sp. 1 gws-like may be too closely related to C. sp. 1 gws to be a distinct species, and could simply reflect the wide range of genetic variation within the species (Figs. 10-22).   Table 7. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.    2.3.3 C. sp. 2 vancouveriensis    Corallina sp. 2 vancouveriensis was also identified in Hind and Saunders (2013A) who reported it as morphologically cryptic with C. vancouveriensis. However, there are still no rbcL sequences for C. sp. 2 vancouveriensis and only one psbA and one CO1 sequence available. Corallina sp. 2 vancouveriensis was sister to C. vancouveriensis in the psbA and CO1 trees (Figs. 12-13), but without branch support. Corallina sp. 2 vancouveriensis appeared distinct from C. vancouveriensis in the psbA and CO1 distance matrices (Table 8). The status of the species cannot be evaluated due to lack of overlapping data.   C. sp. 1 gws  psbA 0 psbA 0.7-1.3CO1 0 CO1 4.5-5.8rbcL / rbcL 1.6-1.9C. sp. 1 gws-like  psbA 0.24 psbA /CO1 3.18 CO1 /rbcL 0.52 rbcL 0C. officinalis  psbA 1.18 psbA 1.41 psbA 0CO1 8.02-8.16 CO1 9.23-9.46 CO1 0rbcL 0.15-0.59 rbcL 2.85-3.24 rbcL 0.15-0.59Hind et al. 2018            C. sp. 1 gwsC. sp. 1 gws-likeC. officinalis  88 Table 8. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.    2.3.4 Taxa surrounding C. ferreyrae E.Y. Dawson, O.C. Acleto, & N. Foldvik   Bustamante (2019) sequenced the Peruvian isotype specimen of C. ferreyrae (Peru, Voucher #UC 1404138), and determined that it was conspecific with Northern Atlantic C. caespitosa (R.H.Walker, J.Brodie & L.M.Irvine). The older binomial name C. ferreyrae has priority over the newer name, C. caespitosa. Bustamante (2019) reported that “A BLAST analysis of cox1, psbA, and rbcL gene markers of C. ferreyrae found exact matches to sequences of C. caespitosa R.H. Walker, J. Brodie, and L.M. Irvine (Walker et al. 2009)”.  However, Walker (2009) only used 18S rRNA and CO1 sequences in their study. While Williamson et al. (2015) later sequenced rbcL, psbA must not have been used in the analyses. Incomplete overlap of data between research groups and a broader definition of “species” in Bustamante et al. (2019) could account for the discrepancy between my study and Bustamante’s findings. I included sequences from Walker et al. (2009) and Bustamante et al. (2019) as well as sequences from other specimens collected from South America (Appendix II, Table S1). Across all three gene trees in my analysis (Figs. 10-15), C. ferreyrae sequences consistently formed a clade with C. sp.1 chile, C. sp. 2 chile, and C. ferreyrae-like, which appear to be four different species in my analysis. C. sp. 2 vancouveriensis  psbA / psbA 0.7-1.3CO1 / CO1 4.5-5.8rbcL / rbcL 1.6-1.9C. vancouveriensis  psbA 0.71-0.71 psbA 0CO1 4.08-4.39 CO1 0-0.45rbcL / rbcL /C. offcinalis  psbA 1.53 psbA 1.18 psbA 0CO1 3.53-4.24 CO1 3.9-4.08 CO1 0rbcL / rbcL 2.74-3.1 rbcL 0.15-0.59C. sp. 2 vancouveriensis Hind et al. 2018         C. vancouveriensisC. officinalis  89 C. sp. 1 chile   Corallina sp. 1 chile was monophyletic with strong branch support across all phylogenetic tree analyses (Figs. 10-22). It was delimited as a species across all three ABGD analyses (Table 2), and exhibited high percent difference from C. ferreyrae and related species with respect to psbA and CO1 gene sequences (Table 9). Interestingly, there was low percent difference between C. sp. 1 chile and other closely related species with respect to rbcL (Table 8). This disagreement between psbA and rbcL is also clearly reflected by psbA and rbcL tree topologies (Figs. 10-11, 14-15). Despite the similarity of rbcL sequences, the majority of evidence, and with a healthy sample size (N=14), strongly supports C. sp. 1 chile as a distinct species and C. sp. 1 chile therefore needs to be given a concrete name, morphological assessment, and described as new to science. This species is a good example of where one might come to a different conclusion if they based their species delimitation on only one gene or only one line of evidence.  C. ferreyrae-like  I applied the provisional name “C. ferreyrae-like” to differentiate specimens that had been previously identified as C. ferreyrae, based on a BLAST search of one gene. Phylogenetically, C. ferreyrae-like consistently formed a sister group to C. ferreyrae sequences with strong branch support for the exclusively C. ferreyrae-like clade (Figs. 10-15). Interestingly, C. ferreyrae was collected from Peru, whereas all C. ferreyrae-like specimens were from Japan. Corallina ferreyrae-like sequences formed a monophyletic group with very strong branch support in all three gene trees (Figs. 10-15). It also formed a clade in the majority rule tree with 67% branch value (Figs. 16-19), an unusually high consistency for this particular genus. Corallina   90 ferreyrae-like was delimited as a species in every ABGD analysis (Table 2) and with a few exceptions was dissimilar from other neighboring species in the distance matrices (Table 9). According to my analyses, C. ferreyrae-like is distinct from C. ferreyrae and may perhaps be correlated with a Japanese type specimen, as all three C. ferreyrae-like specimens (Appendix II, Table S1) were collected in Japan. If not correlated with a type, it should undergo morphological analysis, receive a concrete name, and be described as new to science.  2.3.5 C. sp. 2 chile complex within the C. ferreyrae clade   This complex is composed of provisional species C. sp. 2 chile, the C. caespitosa holotype (Walker et al. 2009), C. melobesioides, and C. pinnatifolia (HQ322333). Corallina caespitosa, which was found to occur in both hemispheres and both the Atlantic and Pacific oceans (Walker et al. 2009), was recently synonymized with the older existing name C. ferreyrae because it corresponded with a Peruvian C. ferreyrae specimen in a genetic analysis (Bustamante et al. 2019). Bustamante et al. (2019) were correct in applying the older name C. ferreyrae for sequences that correlated with the type specimen. However, Bustamante et al. (2019) applied a broad species concept. If the clade containing C. ferreyrae is split into the four narrower species supported in my analysis, then C. caespitosa has to be synonymized under C. pinnatifolia, the oldest name for the C. sp. 2 chile clade (Manza 1937, Dawson 1953).   Especially considering the sample size (N=11), my analyses indicated that the C. sp. 2 chile complex represents at least one distinct species. The majority rule tree (Figs. 16-19) and the concatenated tree (Figs. 20-22) supported C. sp. 2 chile as a clear clade, receiving 88% bootstrap support from the concatenated alignment (Figs. 20-22). Corallina sp. 2 chile formed a clade with strong branch support in the psbA tree (Figs. 10-11). In the CO1 tree however, three clades of   91 sequences of C. sp. 2 chile or C. caespitosa appeared paraphyletic to C. ferreyrae, and C. ferreyrae-like (Figs. 10-11). The paraphyly of C. sp. 2 chile in the CO1 tree seems to reflect biogeography (See Figs. 12-13). That is, the United States C. sp. 2 chile specimen is sister to the C. caespitosa holotype from England, which is in turn sister to a monophyletic strongly supported cluster of three Chilean C. sp. 2 chile specimens. More samples would be required to test for geographical structure in species diversity. Similarly in the rbcL tree, C. sp. 2 chile sequences were likewise split into two clades that were sister to C. sp. 1 chile (Figs. 14-15). Based on distances (Table 9), C. sp. 2 chile is also similar to C. sp.1 chile with respect to rbcL.     Table 9. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.    2.3.6 C. sp. 1 california  Corallina sp. 1 california appeared monophyletic across all phylogenetic analyses, with moderate branch support in the psbA tree (Figs. 10-12), strong support in the CO1 and rbcL trees (Figs. 12-15), and 61 percent bootstrap support in the concatenated tree (Figs. 20-22).   The CO1 ABGD analysis is the only ABGD analysis that grouped C. sp. 1 california independently from other species (Table 2). The psbA ABGD analysis did not resolve C. sp. 1 C. ferreyrae (Bustamante)C. ferreyrae  (Bustamante)  psbA / psbA 0.7-1.3CO1 / CO1 4.5-5.8rbcL / rbcL 1.6-1.9C. ferreyrae-like  psbA 0.82 psbA 0CO1 3.03 CO1 0rbcL 0.82 rbcL 0C. sp. 1 chile  psbA 1.06-1.18 psbA 0.94-1.06 psbA 0-0.12CO1 6.05-6.51 CO1 6.96 CO1 0.15-0.45rbcL 0.97 rbcL 1.2 rbcL 0C. sp. 2 chile  psbA 0.82 psbA 0.94-1.06 psbA 1.06-1.29 psbA 0-0.12CO1 3.03-3.48 CO1 3.03-3.48 CO1 4.39-5.89 CO1 0-1.37rbcL 0.37-0.68 rbcL 0.6-0.75 rbcL 0.6-0.9 rbcL 0.3Hind et al. 2018           C. ferreyrae-likeC. sp. 1 chileC. sp. 2 chile  92 california separately from C. officinalis, C. chilensis, C. sp. 2 frondescens and C. sp. 5 frondescens (Table 2). The rbcL ABGD analysis grouped C. sp. 1 california with C. chilensis and C. sp. 5 frondescens. The distance matrices likewise showed that C. sp. 1 california is similar to these species with respect to psbA and rbcL (Table 10). However, the percent difference between C. sp. 1 california and its closely related species is unusually high with respect to CO1.  Given that C. sp. 1 california forms a well-supported clade across the phylogenetic analyses, it is likely to be a distinct species new to science, despite the lack of resolution in ABGD and percent distance analyses.   2.3.7 C. sp. 4 frondescens   Corallina sp. 4 frondescens sequences formed strongly supported clades across all phylogenetic trees (Figs. 10-22), including with N = 9 sample size in the concatenated tree (Figs. 20-22). It likewise resolved independently in the ABGD analyses (Table 2). The division that appears in the CO1 ABGD analysis and presence of multiple clades in the CO1 phylogeny is likely on account of biogeographical variation (Table 2, Figs. 12-13). Corallina sp. 4 frondescens exhibited high percent difference in at least one gene from all other species to which it was closely related (Table 10). Collectively the evidence in my analyses confirms Hind & Saunders (2013A) recognition of C. sp. 4 frondescens as a distinct species, and it is in need of morphological description and a concrete name.  2.3.8 C. sp. 2 frondescens    Corallina sp. 2 frondescens formed a moderately supported clade in the psbA tree (Figs. 10-11) and a strongly supported clade in the CO1 tree (Figs. 12-13). There was only one sequence for the rbcL gene. The CO1 and rbcL ABGD analyses (Table 2) delimited C. sp. 2   93 frondescens as a distinct species from all other taxa while the psbA ABGD analysis included it in a broader group (Table 2). The difference between C. sp. 2 frondescens and other species was most obvious from the CO1 distance matrix (Table 10). Corallina sp. 2 frondescens was similar to C. sp. 1 california and C. sp. 5 frondescens in the psbA distance matrix (Table 10) and somewhat similar to C. sp. 1 california, C. chilensis, and C. sp. 5 frondescens in the rbcL distance matrix (Fig. 10). While evidence is mixed and more rbcL sequence replicates would be desirable, overall my analyses indicate that C. sp. 2 frondescens is likely an independently evolving population; a distinct species that requires a concrete name and description.  2.3.9 C. sp. 5 frondescens    Corallina sp. 5 frondescens sequences formed a strongly supported clade in the CO1 gene tree (Figs. 12-13). It also appeared to be a distinct clade in the rbcL tree (Figs. 14-15), although it lacked branch support because there was only one sequence (N=1). Corallina sp. 5 frondescens created a well formed clade in the majority rule tree (Figs. 16-19) and in the concatenated tree, but only with 42 percent bootstrap support (Figs. 20-22). The cluster of three C. sp. 5 frondescens sequences in the psbA tree did not have any branch support (Figs. 10-11).   Corallina sp. 5 frondescens was delimited as a separate species only in the CO1 ABGD analysis (Table 2). Corallina sp. 5 frondescens was most distant from other similar species based on the CO1 gene in distance matrices (Fig. 9). Corallina sp. 5 frondescens appears to be an independently evolving population judging by its isolation or branch length in the phylogenetic analyses (Figs. 10-22), but additional evidence is recommended to confirm distinct species status, especially considering the weak support in the concatenated tree and lack of branch support in the psbA and rbcL gene trees.    94 Table 10. Percent differences across all three genes and concatenated analyses. Percent difference ranges from Hind et al. (2018) included for ease of comparison.   2.4 Conclusion  Conclusions regarding the number of species in Corallina may vary across studies depending on the gene or combination of genes used to delimit species, and the type of analyses implemented. For instance, some new species proposed by Hind and Saunders (2013A) were appropriate based solely on CO1 percent difference and the CO1 ABGD analysis, but with more data from psbA and rbcL matrices combined with ABGD analyses, I have expanded the conclusions. ABGD analysis was sensitive to the variation from gene to gene. The most variable locus, CO1, resulted in the narrowest species delimitations. The psbA gene was least variable, and provided the broadest delimitations.   Percent difference ranges of existing published species may be used as guidelines to maintain consistency across studies, but the ranges may also need reevaluation as newer data become available. Some of the proposed species in Hind and Saunders (2013A) were indistinguishable from each other in psbA and rbcL, but differed from other species by >5% in CO1 sequences. If I had just used the CO1 gene to differentiate species, many of the proposed species would be considered distinct based on this single line of evidence, whereas C. C. chilensis  psbA 0-0.24 psbA 0.7-1.3CO1 0.45-0.91 CO1 4.5-5.8rbcL 0.09 rbcL 1.6-1.9C. sp. 1 california  psbA 0.35-0.47 psbA 0CO1 7.41-8.02 CO1 0-0.15rbcL 0.9-1.08 rbcL 0.15C. sp. 2 frondescens  psbA 0.59-0.82 psbA 0.47 psbA 0CO1 6.96-7.26 CO1 6.51-6.66 CO1 0.3rbcL 1.12-1.17 rbcL 1.12-1.27 rbcL /C. sp. 4 frondescens  psbA 0.71-0.94 psbA 0.59 psbA 0.47-0.59 psbA 0CO1 5.9-6.66 CO1 6.51-6.66 CO1 5.9-6.2 CO1 0-1.82rbcL 1.55-1.63 rbcL 1.33-1.55 rbcL 1.63-1.75 rbcL 0.17C. sp. 5 frondescens  psbA 0.24-0.59 psbA 0.12-0.24 psbA 0.35-0.47 psbA 0.12-0.24 psbA 0-0.12CO1 5.9-6.05 CO1 4.99-5.14 CO1 5.14 CO1 4.54-4.69 CO1 0rbcL 0.81 rbcL 0.64-0.73 rbcL 1.05 rbcL 1.13-1.18 rbcL /C. chilensis Hind et al. 2018 C. sp. 1 californiaC. sp. 2 frondescensC. sp. 4 frondescensC. sp. 5 frondescens  95 vancouveriensis would not be considered distinct from C. officinalis (Table 8). If on the other hand I differentiated species by using psbA or rbcL gene analyses and not CO1, C. vancouveriensis would be distinct, but many of the proposed species would be indistinct from one another. This is the reason it is necessary to use multiple genes and multiple lines of evidence when determining species boundaries.   While my study provided additional evidence that confirmed suggestions from previous studies, given the discrepancies and incongruence across analyses, even using three genes and three lines of evidence (phylogenetic analyses, ABGD analyses, percent distance matrices) likely barely scratched the surface with respect to species delimitation. Clearly, there are still many remaining research opportunities within genus Corallina alone, whether confirming existent species or describing new species; and this genus is only one of the many genera in need of taxonomic reform.   For the next steps forward, I first propose an effort to consolidate a small amount of material from as many Corallina type specimens known and available. Ideally DNA could be extracted from as many types as possible, and published so that all research groups could use them to guide the application of names to species. Secondly, I propose obtaining more samples for each species in Corallina from across any given species’ entire known geographic range. Ideally high-throughput sequencing would be used, combined with ecological metadata, and morphological analysis, so that hundreds of loci may be compared using more sophisticated delimitation techniques to create phylogenetic trees that more closely represent actual species trees. Obtaining robust species trees might likewise enable us to identify additional barcoding genes (Zhan et al. 2020). 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How do plastids and mitochondria divide? Microscopy 68(1): 45-56.  Zhan, S.H., Shih, C.C., Liu, S.L. 2020. Reappraising plastid markers of the red algae for phylogenetic community ecology in the genomic era. Ecology and Evolution 10: 1299-1210.  Zasshi, S. 1921. Kichisaburo Yendo. The Botanical Magazine 35(415): 126-130.    109 Appendices  Appendix I: Historical materials   Figure S1. Linnaeus (1758) binomial naming and description of Corallina officinalis.   110  Figure S2. Ellis (1755) description and illustration of what Linnaeus would name “Corallina officinalis.” This is the illustration that Linnaeus cited in his description of C. officinalis in Fig. S1.      Ellis 1755Ellis 48. Plate XXIV  111  Figure S3. Schmitz (1889) designation of Corallina as a genus.    112     Figure S4. Description of Corallina chilensis by Harvey (1849).              Harvey 1847  113  Figure S5. Montagne (1852) report of Corallina chilensis.     114  Figure S6. Ardissone (1888) inventory of species (including Corallina officinalis var. chilensis) in Chiloé, Chile.            115  Figure S7. Yendo (1902A) report of Corallina officinalis var. chilensis.    116     Figure S8. Setchell (1903) report of Corallina officinalis var. chilensis.     Figure S9. Foslie (1907) report of Corallina chilensis.         117  Figure S10. Skottsberg (1923) report of Corallina chilensis.    Skottsberg1923  118  Figure S11. Smith (1944) report of Corallina chilensis.                       Smith1944  119 Appendix II: Sequences Table S1. Table of all sequence data.    * Indicates that corresponding sequence(s) are not in tree, ABGD, or distance matrix analyses.Name Collector# Accession# Justification for designation psbACO1rbcL Location Collector/ ReferencesBossiella frondifera A90727 Published sequence/authority KT782243 KT782032 KT782137 Brady's Blow Hole, Bamfield, Vancouver Island, Canada Hind et al. 2015Calliarthron cheilosporioides GWS010084GenBank BLAST is 100% match with C. cheilosporioides JQ7410 published by van der Merwe et al. 2015.JQ422199 British Columbia, CanadaHind & Saunders 2013A (Note: referred to as "Corallina tuberculosum" in publication and GenBank)Calliarthron cheilosporioides GWS021537 Published sequence/criteria for name application unknown KM254472Point Lobos, California, United States Saunders 2014Calliarthron cheilosporioides NCU585611Published sequence/molecular & morpho comparison of topotype materialHQ322294 Catalina Island, California, United States Gabrielson et al. 2011Chiharaea bodegensis GWS009079Published sequence/morpholgical comparison to topotype materialJQ677009 JQ615596 Wizard I, Bamfield, British Columbia, CanadaHind & Saunders 2013BChiharaea bodegensis GWS010828Published sequence/morphological comparison to topotype materialJQ677000"Most collections were from the Canadian northeast Pacific" (Hind & Saunders 2013)Hind & Saunders 2013BCorallina aberrans PTM 1445 A92983 Clustered with psbA JQ422201 & C01 JQ615597 x x x Katsuura, Japan Patrick T. MartoneCorallina aberrans GWS013777Published sequence morphological comparison to designated lectotype illustrations (Hind & Saunders 2013A)JQ422201 JQ615597 Chibaken, Japan Hind & Saunders 2013ACorallina caespitosa holotype BM000804549 Published sequence/holotype DQ191343 Devon, England Robba et al. 2006; Walker et al. 2009Corallina chilensis PTM 182 A88708 Formed clade with PTM 332 x xBotanical Beach, Vancouver Island, British Columbia, CanadaPatrick T. MartoneCorallina chilensis PTM 332 A89284Identical rbcL sequence with 263 bp Darwin type rbcL sequencex x xHakai, Fifth Beach, Calvert Island, British Columbia, CanadaPatrick T. MartoneCorallina chilensis PTM 738 A91487 Formed clade with PTM 332 xScott's Bay, Bamfield, Vancouver Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 740 A91489 Formed clade with PTM 332 xScott's Bay, Bamfield, Vancouver Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 742 A91491 Formed clade with PTM 332 xScott's Bay, Bamfield, Vancouver Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 743 A91492 Formed clade with PTM 332 xScott's Bay, Bamfield, Vancouver Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 788 A91531 Formed clade with PTM 332 xHakai, Fifth Beach, Calvert Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 789 A91532 Formed clade with PTM 332 xHakai, Fifth Beach, Calvert Island, British Columbia, CanadaKatherine R. HindCorallina chilensis PTM 1244 A92161 Formed clade with PTM 332 x x x Yaquina Head, Oregon, United States Katherine R. HindCorallina chilensis Cor.chi.12ii09* NCU 656905 99.88% similar to PTM 332 over 851 bp in psbA genePlaya Cocholue, Concepcion, Chile (drift) Paul W. GabrielsonCorallina chilensis Remnants from Paul Silva's collection XXXXXIdentical to type specimen (over 263 bp length) x Unknown location, Chile Alcide d'OrbignyCorallina chilensis  type XXXXX Type specimen sequence for C. chilensis (263 bp) x Valparaiso, Chile Charles DarwinCorallina chilensis PTM 789* A91532 Determined by Katherine R. Hind (using DNA)Calvert Island, Fifth Beach, British Columbia, Canada Katherine R. HindCorallina chilensis PTM 487* A89808 Determined by Katherine R. Hind (using DNA)Calvert Island, Fifth Beach, British Columbia, Canada Katherine R. HindCorallina chilensis PTM 182* A88708 Determined by Patrick T. Martone (using DNA)Botanical Beach, Vancouver Island, British Columbia, CanadaPatrick T. MartoneCorallina chilensis PTM 333* A89285 Determined by Katherine R. Hind (using DNA)Calvert Island, Fifth Beach, British Columbia, Canada Patrick T. MartoneCorallina chilensis PTM 629* A89961Determined by Katherine R. Hind and Patrick T. Martone (using DNA)Vancouver Island, Botany Bay, British Columbia, Canada Katherine R. HindCorallina chilensis PTM 326* A89279 Determined by Katherine R. Hind (using DNA)Calvert Island, Fifth Beach, British Columbia, Canada Patrick T. MartoneCorallina chilensis PTM 1588* A93226 Determined by Patrick T. Martone (using DNA)Calvert Island, North Beach Bench, British Columbia, CanadaPatrick T. MartoneCorallina chilensis PTM 1* A88572 100% match to rbcL sequence for PTM 332 over 1107 bpPacific Grove, Hopkins Marine Station, California, USA Patrick T. MartoneCorallina chilensis PTM 10* A88577 100% match to rbcL sequence for PTM 332 over 1107 bpLaguna Beach, Crystal Cove, California USA (drift) Patrick T. MartoneCorallina chilensis PTM 11* A88578 100% match to rbcL sequence for PTM 332 over 1107 bpLaguna Beach, Crystal Cove, California USA (drift) Patrick T. Martone  120   Name Collector# Accession# Justification for designation psbACO1rbcL Location Collector/ ReferencesCorallina crassissima PTM 1447 A92985 Clustered with CO1 JQ615605 x x Katsuura, Japan Patrick T. MartoneCorallina crassissima PTM 1490 A93028 Clustered with psbA JQ422203 x x x Chibaken, Japan Patrick T. MartoneCorallina crassissima GWS013776Published sequence morphological comparison to designated lectotype illustrations (Hind & Saunders 2013A)JQ422203 JQ615605 Chiba prefecture, Katsuura, JapanHind & Saunders 2013ACorallina declinata PTM 1488 A93026 Clustered with psbA JQ422204 & CO1 JQ615613 x x Chibaken, Japan Patrick T. MartoneCorallina declinata GWS013767Published sequence morphological comparison to designated lectotype illustrations (Hind & Saunders 2013A)JQ422204 JQ615613 Chibaken, Japan Hind & Saunders 2013ACorallina ferreyrae PTM 819 A91593Monophyletic with isotype in psbA tree, closest sequence to isotype in CO1 treex x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 821 A91595 Monophyletic with isotype in psbA tree x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 826 A91600 Formed clade with isotype in psbA and rbcL trees x x x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 827 A91601 In clade with isotype in psbA tree x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 830 A91604 In clade with isotype in psbA tree x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 832 A91606 In clade with isotype in psbA tree x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 833 A91607 In clade with isotype in psbA tree x Quintay, Chile Katherine R. HindCorallina ferreyrae PTM 847 A91617 In clade with isotype in psbA tree x Valparaiso, Chile Katherine R. HindCorallina ferreyrae isotype UC1404138 Published sequence/isotype MK408748 MK408747 MK408748 Pucusana, Peru Bustamante et al. 2019Corallina ferreyrae-like PTM 1408 A92946Close sister to C. ferreyrae isotype clade in CO1 & rbcL treesx x Oshoro Bay, Japan, Oshoro Bay Marine Station Patrick T. MartoneCorallina ferreyrae-like PTM 1416 A92954 Formed clade in psbA tree with PTM1440 & PTM 1417 x x x Muroran, Japan Patrick T. MartoneCorallina ferreyrae-like PTM 1417 A92955 Formed clade in psbA tree with PTM1440 and PTM 1416 x Muroran, Japan Patrick T. MartoneCorallina ferreyrae-like PTM 1440 A92978Present in same clade as all other C. ferreyrae-like sequences across all three treesx x x Cape Tachimachi, Hakodate, Japan Patrick T. MartoneCorallina maxima GWS013782Published sequence morphological comparison to designated lectotype illustrations (Hind & Saunders 2013A)JQ422207 JQ615680 Chibaken, Japan Hind & Saunders 2013ACorallina melobesioides UBCa62034 Published sequence/topotype JN701477 Chibaken, Japan Martone et al. 2012Corallina officinalis NCU588445Published sequence/matched other sequences that matched epitypeKJ637651 Alaska, United States Hind et al. 2014 Corallina officinalis GWS006989Published sequence/matched other sequences that matched epitypeJQ422209 Newfoundland & Labrador, CanadaHind & Saunders 2013ACorallina officinalis NCU590595Published sequence/matched other sequences that matched epitypeJQ637652 Foster Island, British Columbia, Canada Hind et al. 2014Corallina officinalis BM001004107Published sequence/matched other sequences that matched epitypeJQ917413 Somerset, England Hind et al. 2014, van der Merwe et al. 2015Corallina officinalis GWS006989 Published sequence/matched with epitype JQ615681Cape Ray, Newfoundland & Labrador, CanadaHind & Saunders 2013ACorallina officinalis NCU588445 Published sequence/in clade with epitype KJ591672Chichigof Harbor, Attu Island, Alaska, United States Hind et al. 2014Corallina officinalis NCU590595 Published sequence/in clade with epitype KJ591674Foster Island, British Columbia,  Canada Hind et al. 2014 Corallina officinalis BM001004107 Published sequence/matched epitype JN701476 Lilstock, Somerset Co., EnglandListed in GenBank as Martone et al. unpublished. Gabrielson must have gotten the sequence or specimen from Brodie, given collector number and location?)Corallina officinalis BM001062598 Published sequence/epitype FM180073 JX315329 Devon, Sidmouth, England Walker et al. 2009;  Brodie et al. 2013  121    Name Collector# Accession# Justification for designation psbACO1rbcL Location Collector/ ReferencesCorallina pinnatifolia UBCa88590 Published representative sequence HQ322333Laguna Beach, Orange County, California, United States Gabrielson et al. 2011Corallina sp. 1 california PTM 363 A89705 Formed clade in psbA tree with JQ422238 x x xHakai, Calvert Island, British Columbia, Canada Sandra LindstromCorallina sp. 1 california PTM 515 A89836 Formed clade in psbA tree with JQ422238 x xHakai, Calvert Island, British Columbia, Canada Sandra LindstromCorallina sp. 1 california PTM 1188 A92117 Formed clade with JQ615736 in CO1 tree x x xHakai, Calvert Island, British Columbia, Canada Patrick T. MartoneCorallina sp. 1 california PTM 1247 A92164 Formed clade with JQ615736 in CO1 tree x x x California, United States Patrick T. MartoneCorallina sp. 1 california GWS021316Published sequence/provisional name sourceJQ422238 JQ615736 Pigeon Point Lighthouse, California, United States Hind & Saunders 2013Corallina sp. 1 chile PTM 862 A91632 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Katherine R. HindCorallina sp. 1 chile PTM 863 A91633 Name designated by Martone lab for group in psbA tree x x x Curinaco, Chile Katherine R. HindCorallina sp. 1 chile PTM 868 A91638 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Katherine R. HindCorallina sp. 1 chile PTM 869 A91639 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Katherine R. HindCorallina sp. 1 chile PTM 876 A91646 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 879 A91649 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 889 A91657 Name designated by Martone lab for group in psbA tree x x Bonifacio, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 891 A91659 Name designated by Martone lab for group in psbA tree x Bonifacio, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 898 A91666 Name designated by Martone lab for group in psbA tree x Bonifacio, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 899 A91667 Name designated by Martone lab for group in psbA tree x x Bonifacio, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 910 A91676 Name designated by Martone lab for group in psbA tree x x x Mar Brava, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 926 A91927 Name designated by Martone lab for group in psbA tree x x Cucao, Chile Patrick T. MartoneCorallina sp. 1 chile PTM 1325 NO VOUCHER Clade with PTM 926 in CO1 & PTM 1337 in rbcL tree x x Cucao, Chile No recordCorallina sp. 1 chile PTM 1337 NO VOUCHER Clase with PTM1325 in rbcL tree x Pucatrihue, Chile No recordCorallina sp. 1 gws PTM 1457 A92995Formed clade with JQ422217 in psbA tree& JQ615738 in the CO1 treex x x Chiba University Marine Institute, Katsuura, Japan Patrick T. MartoneCorallina sp. 1 gws GWS013769Published sequence/provisional name sourceJQ422217 JQ615738 Chibaken, Katsuura, Japan Hind & Saunders 2013Corallina sp. 1 gws-like PTM 1401 A92939Formed clade/sister to C. sp. 1 gws sequences across all three treesx x x Oshoro Bay, Oshoro Marine Station, Japan Patrick T. MartoneCorallina sp. 1 gws-like PTM 1402 A92940 Corresponded with PTM 1401 x Oshoro Bay, Oshoro Marine Station, Japan Patrick T. MartoneCorallina sp. 1 gws-like PTM 1409 A92947 Corresponded with PTM 1401 x Oshoro Bay, Oshoro Marine Station, Japan Patrick T. MartoneCorallina sp. 2 chile PTM 867 A91637 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Katherine R. HindCorallina sp. 2 chile PTM 870 A91640 Name designated by Martone lab for group in psbA tree x x x Curinaco, Chile Katherine R. HindCorallina sp. 2 chile PTM 873 A91643 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Patrick T. MartoneCorallina sp. 2 chile PTM 880 A91650 Name designated by Martone lab for group in psbA tree x Curinaco, Chile Patrick T. MartoneCorallina sp. 2 chile PTM 895 A91663 Name designated by Martone lab for group in psbA tree x x Bonifacio, Chile Patrick T. MartoneCorallina sp. 2 chile PTM 905 A91672 Name designated by Martone lab for group in psbA tree x x Mar Brava, Chile Patrick T. MartoneCorallina sp. 2 chile PTM 1254 A92169 Name designated by Martone lab for group in psbA tree x x xArrowhead point, Stillwater cove Pebble Beach CA Patrick T. MartoneCorallina sp. 2 chile PTM 1262 NO VOUCHER Name designated by Martone lab for group in psbA tree xArrowhead point, Stillwater cove Pebble Beach CA No recordCorallina sp. 2 chile PTM 1265 A92173 Name designated by Martone lab for group in psbA tree xArrowhead point, Stillwater cove Pebble Beach CA Patrick T. MartoneCorallina sp. 2 chile PTM 1266 A92174 Name designated by Martone lab for group in psbA tree xArrowhead point, Stillwater cove Pebble Beach CA Patrick T. MartoneCorallina sp. 2 chile PTM 1519 A93057 Name designated by Martone lab for group in psbA tree xEast of Shimen Harbour, Keelung, Taiwan Patrick T. MartoneCorallina sp. 2 chile PTM 1985* XXXXX Field identified as "C. officinalis var. chilensis" (x) Biobio, Chile Erasmo MacayaCorallina sp. 2 chilePC0028646, or PC0028647, or PC0040576Clustered with C. sp. 2 chile specimens in rbcL type tree (263 bp)x "San Carlos De Chiloe" (Ancud), Chile, Collected year 1836. Claudio Gay  122  Name Collector# Accession# Justification for designation psbACO1rbcL Location Collector/ ReferencesCorallina sp. 2 frondescens PTM 489 A89810 Formed clade with PTM 1178 in psbA tree xHakai, Calvert Island, British Columbia, Canada Katherine R. HindCorallina sp. 2 frondescens PTM 1178 A92108 Formed clade with JQ615748 in CO1 tree x x xHakai, North Beach, Calvert Island, British Columbia, CanadaPatrick T. MartoneCorallina sp. 2 frondescens GWS003062Published sequence/provisional name sourceJQ615748Seapool Rock, Bamfield, Vancouver Island, British Columbia, CanadaHind & Saunders 2013Corallina sp. 2 vancouveriensis GWS009913Published sequence/provisional name sourceJQ422229 JQ615760Tahsis, Island #40 on Esperenza Inlet Chart, British Columbia, CanadaHind & Saunders 2013Corallina sp. 3 frondescens PTM 1400 A92938 Formed clade with JQ2221 in psbA tree x x xOshoro Bay, Japan, Oshoro Bay Marine Station Patrick T. MartoneCorallina sp. 3 frondescens PTM 1405 A92943 Formed clade with JQ2221 in psbA tree x x xOshoro Bay, Japan, Oshoro Bay Marine Station Patrick T. MartoneCorallina sp. 3 frondescens PTM 1442 A92980 Formed clade with JQ2221 in psbA tree x x xCape Tachimachi, Hakodate, Japan Patrick T. MartoneCorallina sp. 3 frondescens GWS006466Published sequence/provisional name sourceJQ22221 JQ615765 Stephenson Pt., British Columbia, Canada Hind & Saunders 2013Corallina sp. 3 frondescens GWS011941Published sequence/provisional name sourceJQ615766 Hokkaido University Marine Station, Oshoro Bay, Japan Hind & Saunders 2013Corallina sp. 3 frondescens-like PTM 1419 A92957Formed clade sister to C. sp. 3 frondescens x x Muoran, Japan Patrick T. MartoneCorallina sp. 3 frondescens-like PTM 1439 A92977Formed clade sister to C. sp. 3 frondescens x x x Cap Patrick T. MartoneCorallina sp. 4 frondescens PTM 822 A91596 Formed clade with JQ422222 in psbA tree xOshoro Bay, Japan, Oshoro Bay Marine Station Katherine R. HindCorallina sp. 4 frondescens PTM 842 A91612 Formed clade with JQ422222 in psbA tree x Valparaiso Torpederas Chile Katherine R. HindCorallina sp. 4 frondescens PTM 844 A91614 Formed clade with JQ422222 in psbA tree x x x Valparaiso Torpederas Chile Katherine R. HindCorallina sp. 4 frondescens PTM 846 A91616 Formed clade with JQ422222 in psbA tree x x Valparaiso Torpederas Chile Katherine R. HindCorallina sp. 4 frondescens PTM 881 A91651 Formed clade with JQ422222 in psbA tree x x Curinaco, Chile Patrick T. MartoneCorallina sp. 4 frondescens PTM 1235 NO ACCESSION Formed clade with JQ422222 in psbA tree x xSeal rocks state park beach, Oregon USA No recordCorallina sp. 4 frondescens GWS010351Published sequence/provisional name sourceJQ422222 British Columbia, Canada Hind & Saunders 2013Corallina sp. 4 frondescens GWS021267Published sequence/provisional name sourceJQ615770 Pigeon Point Lighthouse, California, United States Hind & Saunders 2013Corallina sp. 4 frondescens GWS010351Published sequence/provisional name sourceJQ615787 Point Holmes, Comox, British Columbia, Canada Hind & Saunders 2013Corallina sp. 5 frondescens PTM 420 A89741Fell into clad with other C. sp. 5 frondescens in psbA & CO1 treesx x xHakai, Wolf Beach, Calvert Island, British Columbia, CanadaKatherine R. HindCorallina sp. 5 frondescens GWS006561Published sequence/provisional name sourceJQ422226 JQ615794 Tahsis, Princesa Channel, British Columbia, Canada Hind & Saunders 2013Corallina sp. 5 frondescens GWS012660Published sequence/provisional name sourceJQ422227 HM918986Mazarredo Islands, NW of Masset, Haida Gwaii, British Columbia, CanadaHind & Saunders 2013Corallina sp. 5 Korea GWS018201Published sequence/provisional name sourceJQ615795 Lighthouse Point, Piyangdo Island, South Korea Hind & Saunders 2013Corallina vancouveriensis PTM 760 A91506 Compared to topotype specimens x xHakai, Calvert Island, British Columbia, Canada Katherine R. HindCorallina vancouveriensis PTM 767 A91513 Compred to topotype specimens x x xHakai, Calvert Island, British Columbia, Canada Katherine R. HindCorallina vancouveriensis GWS010831 Published sequence/compared with topotype JQ422228 JQ615834Seppings I, Bamfield, British Columbia, Canada Hind & Saunders 2013Corallina vancouveriensis PTM 179* A88705Determined by Patrick T. Martone/comparison to topotypeBotanical Beach, Vancouver Island, British Columbia, CanadaPatrick T. MartoneCrusticorallina muricata UBCa89963 Published sequence/authority KU983300 Botany Bay, Vancouver Island, British Columbia, Canada Hind et al. 2016 Crusticorallina muricata UBCa91387 Published sequence/authority KU983192Brady's Beach Blowhole, Bamfield, Vancouver Island, British Columbia, CanadaHind et al. 2016 Crusticorallina muricata UBCa89963 Published sequence/authority KU983253 Botany Bay, Vancouver Island, British Columbia, Canada Hind et al. 2016 Ellisolandia elongata GWS001818 Published sequence/authority JQ422231 Leitrim, Ireland Hind & Saunders 2013Ellisolandia elongata GWS001818 Published sequence/authority JQ615843 Leitrim, Ireland Hind & Saunders 2013Ellisolandia elongata BM000806006 Published sequence/criteria for name application unknown KP834400 Llanes, Asturias, Spain Williamson et al. 2015Lithothamnion glaciale GWS007312 Published sequence/criteria for name application unknown KP224290Newfoundland & Labrador, Maerl bed, Canada Hind et al. 2018Lithothamnion glaciale none given Published sequence/criteria for name application unknown HM918805Newfoundland & Labrador, CanadaHind et al. 2018; iBOL data release, 2018Lithothamnion glaciale GWS007312 Published sequence/criteria for name application unknown KC134336Newfoundland & Labrador, Maerl bed, Canada Hind et al. 2018Genus that has yet to be described PTM 1984* XXXXXField identified as "C. officinalis var. chilensis" (x) Biobio, Chile Erasmo Macaya  123      Table S2. Concatenated outgroup sequences GenBank numbers for concatenated tree.      Table S3. Table of (N=91) Corallina chilensis specimens collected by Hind & Saunders (2013A), corresponding GenBank sequence numbers, and locations.   Name psbA CO1 rbcLLithothamnion glaciale KP224290 HM918805 KC134336Calliarthron cheilosporioides JQ422199 KM254472 HQ322294Chiharaea bodegensis JQ677009 JQ615596 JQ677000Bossiella frondifera KT782243 KT782032 KT782137Ellisolandia elongata JQ422231 JQ615843 KP834400Crusticorallina muricata KU983300 KU983192 KU9832531GenBank# BLAST Results Species determination Collection # Province/State Country lat longHM918990 Matched 100% with HQ545178 CO1 Corallina chilensis GWS012704 British Columbia Canada 54.111 -132.37HM919003 Matched 100% with HQ544623 CO1 Corallina chilensis GWS012926 British Columbia Canada 52.446 -131.23HM919004 CO1 99.39% match to PTM 332 Corallina chilensis GWS012933 British Columbia Canada 52.446 -131.23HQ544551 CO1 99.39% match to PTM 332 Corallina chilensis GWS019642 British Columbia Canada 48.838 -125.13HQ544596 CO1 99.70% match to PTM 332 Corallina chilensis GWS019732 British Columbia Canada 52.442 -131.32HQ544623 CO1 100% match PTM 332 Corallina chilensis GWS019794 British Columbia Canada 52.442 -131.32HQ544655 CO1 100% match with HQ544623 Corallina chilensis GWS019852 British Columbia Canada 52.442 -131.32HQ544681 CO1 100% match with HQ544623 Corallina chilensis GWS019949 British Columbia Canada 52.428 -131.38HQ544693 CO1 100% match with JQ615658 Corallina chilensis GWS019973 British Columbia Canada 52.433 -131.37HQ544765 CO1 100% match with HQ545178 Corallina chilensis GWS020126 British Columbia Canada 52.45 -131.29HQ544777 CO1 100% match with HQ545178 Corallina chilensis GWS020165 British Columbia Canada 52.45 -131.29HQ544839 CO1 99.24% match to PTM 332 Corallina chilensis GWS020272 British Columbia Canada 52.358 -131.17HQ544903 CO1 100% match to HQ545178 Corallina chilensis GWS020417 British Columbia Canada 52.578 -131.44HQ544917 CO1 100% match with HQ545178 Corallina chilensis GWS020436 British Columbia Canada 52.578 -131.44HQ544937 CO1 100% match with HQ544623 Corallina chilensis GWS020482 British Columbia Canada 52.579 -131.44HQ544993 CO1 100% match with HQ545178 Corallina chilensis GWS020579 British Columbia Canada 52.762 -131.61HQ545000 CO1 99.19% match to PTM 332 Corallina chilensis GWS020590 British Columbia Canada 52.762 -131.61HQ545012 CO1 100% match to HQ545178 Corallina chilensis GWS020614 British Columbia Canada 54.033 -132.05HQ545022 CO1 100% match to HQ545178 Corallina chilensis GWS020648 British Columbia Canada 54.033 -132.05HQ545055 CO1 100% mach to HQ545178 Corallina chilensis GWS020744 British Columbia Canada 53.217 -131.99HQ545057 CO1 100% match to HQ545174 Corallina chilensis GWS020746 British Columbia Canada 53.217 -131.99HQ545067 CO1 100% match to HQ545174 Corallina chilensis GWS020761 British Columbia Canada 53.217 -131.99HQ545119 CO1 100% match to HQ545178 Corallina chilensis GWS020834 British Columbia Canada 53.242 -132.02HQ545129 CO1 100% match to HQ545178 Corallina chilensis GWS020848 British Columbia Canada 53.248 -131.98HQ545147 CO1 100% match to HQ545178 Corallina chilensis GWS020874 British Columbia Canada 53.248 -131.98HQ545174 CO1 99.54% match to PTM 332 Corallina chilensis GWS020910 British Columbia Canada 53.248 -131.98HQ545178 CO1 99.24% match to PTM 332 Corallina chilensis GWS020917 British Columbia Canada 53.248 -131.98HQ545198 CO1 100% match to HQ545178 Corallina chilensis GWS020952 British Columbia Canada 54.107 -132.37HQ545202 CO1 100% match to HQ545178 Corallina chilensis GWS020957 British Columbia Canada 54.107 -132.37HQ545209 CO1 100% match to HQ545178 Corallina chilensis GWS020965 British Columbia Canada 54.107 -132.37JQ615617 CO1 99.24% match to PTM 332 Corallina chilensis GWS013274 British Columbia Canada 52.604 -131.45JQ615619 CO1 99.54% match to PTM 332 Corallina chilensis GWS010231 British Columbia Canada 49.821 -126.98JQ615620 CO1 100% match to HQ545178 Corallina chilensis GWS010230 British Columbia Canada 49.821 -126.98JQ615621 CO1 100% match to HQ544551 Corallina chilensis GWS010119 British Columbia Canada 49.609 -126.61JQ615622 CO1 100% match to HQ545178 Corallina chilensis GWS010026 British Columbia Canada 49.813 -126.99JQ615623 CO1 100% match to HQ545178 Corallina chilensis GWS010020 British Columbia Canada 49.813 -126.99JQ615624 CO1 100% match to HQ544551 Corallina chilensis GWS009931 British Columbia Canada 49.813 -126.99JQ615625 CO1 99.24% match to PTM 332 Corallina chilensis GWS009929 British Columbia Canada 49.813 -126.99JQ615626 CO1 100% match to HQ545178 Corallina chilensis GWS009923 British Columbia Canada 49.813 -126.99JQ615627 CO1 99.21% match to PTM 332 Corallina chilensis GWS009920 British Columbia Canada 49.813 -126.99JQ615628 CO1 100% match to JQ615625 Corallina chilensis GWS009911 British Columbia Canada 49.813 -126.99  124       1GenBank# BLAST Results Species determination Collection # Province/State Country lat longJQ615629 CO1 99.39% match to PTM 332 Corallina chilensis GWS009910 British Columbia Canada 49.813 -126.99JQ615630 CO1 99.39% match to PTM 332 Corallina chilensis GWS009698 British Columbia Canada 49.813 -126.99JQ615631 CO1 99.09% match to PTM 332 Corallina chilensis GWS009695 British Columbia Canada 49.813 -126.99JQ615632 CO1 100% match to JQ615647 Corallina chilensis GWS008233 British Columbia Canada 48.824 -125.16JQ615633 CO1 100% match to HQ545178 Corallina chilensis GWS006658 British Columbia Canada 49.813 -126.99JQ615634 CO1 99.24% match to PTM 332 Corallina chilensis GWS009442a British Columbia Canada 48.352 -123.73JQ615635 CO1 100% match to HQ545178 Corallina chilensis GWS010755 British Columbia Canada 48.852 -125.12JQ615636 CO1 100% match to HQ545178 Corallina chilensis GWS010754 British Columbia Canada 48.852 -125.12JQ615637 CO1 100% match to HQ545178 Corallina chilensis GWS010753 British Columbia Canada 48.852 -125.12JQ615638 CO1 98.94% match to PTM 332 Corallina chilensis GWS010751 British Columbia Canada 48.852 -125.12JQ615639 CO1 100% match to HQ545178 Corallina chilensis GWS010742 British Columbia Canada 48.852 -125.12JQ615640 CO1 100% match to HQ545178 Corallina chilensis GWS010634 British Columbia Canada 48.835 -125.15JQ615641 CO1 100% match to JQ615631 Corallina chilensis GWS004343 British Columbia Canada 48.53 -124.45JQ615642 CO1 100% match to HQ545178 Corallina chilensis GWS002859 British Columbia Canada 48.858 -125.16JQ615643 CO1 100% match to HQ544839 Corallina chilensis GWS002818 British Columbia Canada 48.852 -125.12JQ615644 CO1 100% match to HQ544839 Corallina chilensis GWS001450 British Columbia Canada 48.824 -125.16JQ615645 CO1 100% match to HQ545178 Corallina chilensis GWS008204 British Columbia Canada 48.824 -125.16JQ615646 CO1 100% match to HQ544551 Corallina chilensis GWS004885 British Columbia Canada 54.234 -130.8JQ615647 CO1 99.39% match to PTM 332 Corallina chilensis GWS010302 British Columbia Canada 49.746 -126.64JQ615648 CO1 100% match to HQ545178 Corallina chilensis GWS010267 British Columbia Canada 49.725 -126.64JQ615649 CO1 100% match to JQ615647 Corallina chilensis GWS010264 British Columbia Canada 49.725 -126.64JQ615650 CO1 100% match to JQ615647 Corallina chilensis GWS010259 British Columbia Canada 49.725 -126.64JQ615651 CO1 98.79% match to PTM 332 Corallina chilensis GWS022305 California United 36.592 -121.96JQ615652 CO1 100% match to JQ615647 Corallina chilensis GWS021315 California United 37.183 -122.39JQ615653 CO1 100% match to JQ615647 Corallina chilensis GWS021298 California United 37.183 -122.39JQ615654 CO1 100% match to HQ544839 Corallina chilensis GWS021241 California United 37.183 -122.39JQ615655 CO1 99.09% match to PTM 332 Corallina chilensis GWS011047 British Columbia Canada 49.213 -123.94JQ615656 CO1 100% match to JQ615625 Corallina chilensis GWS011052 British Columbia Canada 49.213 -123.94JQ615657 CO1 99.24% match to PTM 332 Corallina chilensis GWS013075 British Columbia Canada 52.586 -131.37JQ615658 CO1 99.24% match to PTM 332 Corallina chilensis GWS013603 British Columbia Canada 52.462 -131.45JQ615659 CO1 100% match with JQ615658 Corallina chilensis GWS013609 British Columbia Canada 52.462 -131.45JQ615660 CO1 99.70% match to PTM 332 Corallina chilensis GWS013610 British Columbia Canada 52.462 -131.45JQ615661 CO1 100% match with JQ615660 Corallina chilensis GWS013615 British Columbia Canada 52.462 -131.45JQ615662 CO1 100% match with JQ615657 Corallina chilensis GWS013659 British Columbia Canada 52.433 -131.37JQ615663 CO1 99.70% match to PTM 332 Corallina chilensis GWS013661 British Columbia Canada 52.433 -131.37JQ615664 CO1 99.09% match to PTM 332 Corallina chilensis GWS012563 British Columbia Canada 53.152 -132.59JQ615665 CO1 99.39% match to PTM 332 Corallina chilensis GWS012945 British Columbia Canada 52.446 -131.23JQ615666 CO1 100% match to HQ545178 Corallina chilensis GWS013076 British Columbia Canada 52.586 -131.37JQ615667 CO1 100% match to HQ545178 Corallina chilensis GWS013242 British Columbia Canada 52.575 -131.44JQ615668 CO1 100% match to HQ545178 Corallina chilensis GWS013272 British Columbia Canada 52.604 -131.45JQ615669 CO1 100% match to HQ545178 Corallina chilensis GWS013275 British Columbia Canada 52.604 -131.45JQ615670 CO1 100% match to HQ545178 Corallina chilensis GWS013276 British Columbia Canada 52.604 -131.45JQ615671 CO1 100% match to HQ545178 Corallina chilensis GWS013281 British Columbia Canada 52.604 -131.45JQ615672 CO1 100% match to HQ545178 Corallina chilensis GWS013284 British Columbia Canada 52.604 -131.45JQ615673 CO1 100% match to JQ615664 Corallina chilensis GWS013286 British Columbia Canada 52.604 -131.45JQ615674 CO1 100% match to HQ545178 Corallina chilensis GWS013287 British Columbia Canada 52.604 -131.45JQ615675 CO1 98.94% match to PTM 332 Corallina chilensis GWS002775 British Columbia Canada 48.852 -125.12JQ615676 CO1 100% match to HQ545178 Corallina chilensis GWS013613 British Columbia Canada 52.462 -131.45JQ615677 CO1 100% match to HQ545178 Corallina chilensis GWS013614 British Columbia Canada 52.462 -131.45JQ615678 CO1 99.09% match to PTM 332 Corallina chilensis GWS013657 British Columbia Canada 52.433 -131.37  125  Figure S12. Corallina chilensis collected from the Northeast Pacific sites north of Oregon. Green dots indicate PTM collection sites for specimens included in morphometric analysis (Table S4), blue dots indicate Hind & Saunders collection sites (Hind & Saunders 2013A, see Table S3).                         126 Appendix III: Morphological measurements      Table S4. Corallina chilensis measurements for morphological analysis.                1Species PTM Herbarium# grounds for name Country Lat Long Random frond width (mm)Random frond length (mm)Crown length (mm)Stem length (mm)Main axis mid intergen. max width (um)Main axis mid intergen. min width (um)Main axis mid intergen. length (um)Basal intergen. width (um)Basal intergen. length (um)Branch intergen. max width (um)Branch intergen. min width (um)Branch intergen. length (um)Full branch length (mm)Concept. width (um)Concept. length (um)Tallest frond width (mm)Tallest frond length (mm)C. chilensis 182 A88708 Strong clade with PTM 332 & PTM 1244 in psbA & CO1 trees.  PTM 332 & 1244 rbcL matches with Darwin 2151 in rbcL tree.  (PTM 182 matches 99.58% in psbA to UC2050474 in NCBI BLASTn)Canada 48.529253 -124.453704 40.09 67.68 67.68 0 1862 1440 1922 1367 2113 1331 771.5 1512 20.49 700.2 1339 34.87 115.31C. chilensis 209 A89561 100% rbcL BLAST match with UC2050474, (UC2050474 matched PTM182)Canada 51.651533 -128.146583 20.39 50.09 21.7 28.39 1620 785.9 1263 1060 691.8 890.5 606.6 1940 18.79 56.18 57.2C. chilensis 306 A89266 99.84% rbcL BLAST match with UC2050474 Canada 51.66454 -128.1347 25.07 39.18 23.01 16.17 1859 816.7 1834 1517 2319 1127 474.6 1565 17.53 43.13 57.55C. chilensis 311 A89662 99.52% rbcL BLAST match with UC2050474 Canada 51.651533 128.146583 15.83 40.42 25.73 14.69 1818 1092 1622 1084 1234 1428 779.2 2023 8.43 15.83 40.42C. chilensis 326 A89279 99.71% rbcL BLAST match with UC2050474 Canada 51.64358056 -128.15815 46.96 66.24 45.52 20.72 2019 1048 1702 1139 740.3 1515 37.92 54.04 71.83C. chilensis 332 A89284 Supported by psbA, CO1, & rbcL trees Canada 51.64358056 -128.15815 37.09 43.95 38.85 5.1 1760 1170 1337 1460 1243 1235 468.9 1776 16.77 37.09 43.95C. chilensis 333 A89285 99.17% rbcL BLAST match with UC2050474 Canada 51.64358056 -128.15815 29.01 35.17 35.17 0 2042 1162 1458 1830 1387 1949 1065 1585 29.2 17.76 47.15C. chilensis 335 A89286 99.2% rbcL BLAST match with UC2050474 Canada 51.64358056 -128.15815 14.68 29.32 19.01 10.31 1580 891.1 1460 824.4 739.3 1330 576.1 1377 8.45 27.85 38.4C. chilensis 337 A89288 99.76% rbcL BLAST match with UC2050474 Canada 51.64358056 -128.15815 30.24 54.86 54.86 0 1509 895.1 1459 1348 1468 1387 705.6 1722 11.65 30.24 54.86C. chilensis 362 A89704 99.54% rbcL BLAST match with UC2050474 Canada 51.66545 -128.136033 21.42 32.76 20.49 12.27 1490 872.3 1321 1604 1054 1447 630.5 1408 8.79 22.09 42.83C. chilensis 487 A89808 99.57% psbA BLAST match with UC2050474 Canada 51.64358 -128.1581667 21.21 48.99 22.91 26.08 1922 1302 2409 1191 848.3 2488 1026 1965 12.7 51.52 54.16C. chilensis 629 A89961 99.67% psbA BLAST match with UC2050474 Canada 48.52959 -124.45472 51.17 95.2 87.2 8 1493 732.2 1503 1112 872.2 1244 702.7 1366 33.97 664.3 958.8 60.1 93.91C. chilensis 726 A91477 98.47% psbA BLAST match with UC2050474 Canada 48.82432778 -125.1610139 17.22 18.23 18.23 0 1380 781.9 1254 1183 1084 1096 408.6 1165 7.13 17.3 26.33C. chilensis 738 A91487 Supported by psbA tree Canada 48.8341 -125.1456194 15.85 14.24 11.08 3.16 1627 921.8 1424 1239 1574 997.2 602.5 1579 6.49 615.7 1289 11.82 15.98C. chilensis 740 A91489 Supported by psbA tree Canada 48.8341 -125.1456194 15.57 34.87 34.87 0 1676 854.7 1548 1167 1057 1585 622.4 1613 11.63 627.9 1557 12.14 36.72C. chilensis 742 A91491 Supported by psbA tree Canada 48.8341 -125.1456194 23.55 31.15 31.15 0 2049 1288 1697 1666 1564 1417 677.6 1629 19.27 705.2 1723 25.25 30.85C. chilensis 743 A91492 Supported by psbA tree Canada 48.8341 -125.1456194 15.13 17.13 17.13 0 1677 1009 1262 1619 1203 1304 592.4 1358 6.62 615 1216 7.19 24.2C. chilensis 763 A91509 98.86% psbA BLAST match with UC2050474 Canada 51.66453889 -128.1347 21 42.98 32.34 10.64 1096 614.6 1644 887 1653 1446 645.6 1868 8.4 29 54.13C. chilensis 788 A91531 Supported by psbA tree Canada 51.64358056 -128.15815 31.56 54.86 31.1 23.76 1365 910.7 1229 752.2 871.5 1221 529.6 1552 16.34 604.8 2544 30.34 79.03C. chilensis 789 A91532 Supported by psbA tree Canada 51.64358056 -128.15815 14.24 48.35 23.73 24.62 1338 888.8 1507 689.9 1211 979.2 671.2 1804 10.8 31.79 63.28C. chilensis 975 A91962 99.57% psbA BLAST match with UC2050474 Canada 51.66454 -128.1347 8.97 25.65 17.79 7.86 1075 841.3 1825 1066 1142 2159 993.4 1814 7.39 17.21 41.95C. chilensis 1244 A92161 Supported psbA, CO1, & rbcL trees USA 44.67526 -124.07826 10.15 17.79 12.79 5 1330 1015 1676 1515 1642 1218 661.8 1936 9.32 7.3 22.83*UC2050474 corresponds with NC_042901 and MK598845 in GenBank.  The specimen is from Tomales, California.   Publication: Alejo et al. 2019  127 Table S5. Corallina vancouveriensis measurements for morphological analysis.   1PTM# Herbarium# Species Reason for determination Country Lat Long Intergeniculum* max width (um)Intergeniculum*min width (um)Intergeniculum* length (um)Conceptacle width (um)Random frond length (mm)5 A88574 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 36.6217444 -121.9057944 881.7 506.9 618.1 425.9 44.7312 A88579 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 33.574153 -117.843647 650.4 366.3 660 445.8 44.496 A88624 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -122.770666 824.3 551.7 942.9 493.7 47.44156 A88682 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 791.8 503.9 958.2 579.5 61.66158 A88684 vancouveriensis In Genbank:  KJ637656  Hind et al. 2014Canada 48.529253 -124.453704 964.1 542.9 943.9 581.6 72.62161 A88687 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 776.6 498.2 821.9 471.2 70.42163 A88689 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 1214 674.2 966.8 590.4 53173 A88699 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 998.3 404.1 841.6 556 48.96177 A88703 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 888.2 454 675.4 496 52.69179 A88705 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 48.529253 -124.453704 1102 565.8 1095 56.4212 A89564 vancouveriensis Determined genetically or morphologically by PTM & KRHCanada 51.651533 -128.1347 862.8 533.5 821.6 586.7 64.2313 A89448 vancouveriensis 99.10% rbcL BLAST match with NCU588197/HQ322334 Gabrielson et al. 2011Canada 766.4 456.5 936.6 377.5 71.55320 A89274 vancouveriensis 100% rbcL BLAST match NCU588197/HQ322334 Gabrielson et al. 2011Canada 627.5 433.2 807.8 462.4 60.36328 A89280 vancouveriensis 99.71% rbcL BLAST match HQ3223341/NCU588197 Gabrielson et al. 2011Canada 708.1 442.9 889.1 461.6 61.01714 A91468 vancouveriensis 98.63% BLAST psbA match to UBCA88684/KJ637656 Hind et al. 2014Canada 48.8243278 -125.1610139 848.3 492.7 657.1 442.9 34.61715 A91469 vancouveriensis 98.01% psbA BLAST match to A88684/KJ637656 Hind et al. 2014Canada 48.8243278 -125.1610139 768.8 410.2 715.5 433.1 39.88758 A91504 vancouveriensis 100% psbA BLAST match to JQ422228 & KJ637656 Hind & Saunders 2013A/UBC A88684 Hind et al. 2014Canada 51.6645389 -128.1347 604.8 380.2 834.1 472.3 46.23760 A91506 vancouveriensis psbA tree in clade ith JQ422228 Canada 51.6645389 -128.1347 806.8 678.7 384 695 30.72767 A91513 vancouveriensis psbA tree in clade ith JQ422228, CO1 tree with JQ615834, in rbcL treeCanada 51.6645389 -128.1347 851.4 538.6 801.1 538.9 45.36*Intergeniculum located middle of main axis  128 Appendix IV: Methods tables  Table S6. Thermal cycler settings.      Table S7. Rates of evolution and models for tree analyses.      psbA 20 ul Temp ( C ) Time Step psbA 25 ul Temp ( C ) Time CycleInitial denaturation 94 4:00 1 Initial denaturation 94 5:00 1Denaturation 94 1:00 2 Denaturation 94 0:30 2Annealing 50 0:30 3 Annealing 50 0:30 3Extension 72 1:00 4 Extension 72 0:42 4Go to step 2 30x 5 Go to step 2 30x 5Final extension 72 7:00 6 Final extension 72 7:00 6CO1 20 ul Temp ( C ) Time Step CO1 25 ul Temp ( C ) Time CycleInitial denaturation 94 2:00 1 Initial denaturation 94 5:00 1Denaturation 94 0:30 2 Denaturation 94 0:10 2Annealing 45 0:30 3 Annealing 46.5 0:20 3Extension 72 1:00 4 Extension 72 0:30 4Go to step 2 5x 5 Go to step 2 40x 5Denaturation 94 0:30 6 Final extension 7:00 6Annealing 46.5 0:30 7Extension 72 1:00 8Go to step 6 35x 9Final extension 72 7:00 10rbcL 20 ul Temp ( C ) Time Step rbcL 25 ul Temp ( C ) Time CycleInitial denaturation 95 2:00 1 Initial denaturation 95 5:00 1Denaturation 93 1:00 2 Denaturation 93 0:10 2Annealing 47 1:00 3 Annealing 47 0:20 3Extension 72 2:00 4 Extension 72 0:30 4Go to step 2 35x 5 Go to step 2 40x 5Final extension 72 2:00 6 Final extension 72 7:00 6Alignment Gene Codon Iqtree MAC aLRT/MLMrBayes "set models" Partition mymodels on PC MrBayespsbA psbA 1 TNe+Inst=6 rates=propinv; statefreqpr=fixed(equal) SYM+IpsbA 2 F81+F+I nst=1 rates=propinv F81+F+IpsbA 3 HKY+F+G4 nst=2 rates=gamma HKY+F+G4CO1 CO1 1 TN+F+G4 nst=6 rates=gamma GTR+F+G4CO1 2 F81+F+I nst=1 rates=propinv F81+F+ICO1 3 K3Pu+F+I+G4 nst=1 rates=propinv GTR+F+I+G4rbcL rbcL 1 TIM+F+I nst=6 rates=propinv GTR+F+IrbcL 2 F81+F+I nst=1 rates=propinv F81+F+IrbcL 3 TIM3+F+G4 nst=6 rates=gamma  GTR+F+G4rbcL type rbcL 1 TIM+F+I nst=6 rates=propinv GTR+F+IrbcL 2 F81+F+I nst=1 rates=propinv F81+F+IrbcL 3 TIM3+F+G4 nst=6 rates=gamma GTR+F+G4  129 Appendix V: Distance matrices     Table S8. Percent difference matrix of psbA sequences.          C. sp. 1 chileC. sp. 1 gwsC. sp. 2 chileC. aberransC. sp. 1 californiaC. officinalisC. crassissimaC. vancouveriensisC. declinataC. ferreyrae Lithothamnion glacialeC. ferreyrae-likeC. gws-likeC. sp. 3 frondescens-likeC. maximaBossiella frondiferaCalliarthron cheilosporioidesChiharaea bodegensisCrusticorallina muricataEllisolandia elongataC. chilensisC. sp. 2 frondescensC. sp. 2 vancouveriensisC. sp. 3 frondescensC. sp. 4 frondecensC. sp. 5 frondescens0.12 1.18 1.29 1.41 0.82 0.94 1.53 1.65 1.29 1.18 10.34 1.06 1.29 1.76 1.41 6.70 6.51 6.68 4.94 6.00 1.29 1.06 2.00 1.65 1.41 1.060.00 1.06 1.06 1.18 0.71 0.82 1.41 1.53 1.06 0.94 10.22 0.94 1.18 1.65 1.29 6.58 6.39 6.56 4.82 5.88 1.06 0.94 1.88 1.41 1.29 0.820.00 1.76 1.65 1.06 1.18 1.76 1.41 1.53 1.41 9.99 1.53 0.24 2.00 1.06 6.23 6.63 6.43 4.70 6.13 1.41 1.18 2.12 1.88 1.53 1.180.00 1.65 1.53 1.06 1.18 1.76 1.41 1.41 1.30 9.99 1.53 0.24 2.00 1.06 6.23 6.63 6.43 4.70 6.13 1.29 1.18 2.12 1.76 1.53 1.060.12 1.53 0.94 1.06 1.65 1.53 1.41 0.82 9.64 1.06 2.00 1.88 1.76 6.58 6.88 6.68 5.05 6.00 1.41 1.18 1.88 1.76 1.18 1.180.00 1.29 0.82 0.94 1.53 1.41 1.29 0.59 9.52 0.94 1.88 1.76 1.65 6.46 6.76 6.56 4.94 5.88 1.18 1.06 1.76 1.53 1.06 0.940.12 0.82 0.94 0.59 1.41 0.12 1.18 9.64 1.53 1.88 1.29 1.41 5.41 6.27 5.53 4.47 5.88 1.29 0.82 1.76 1.18 1.18 1.060.12 0.71 0.82 0.47 1.29 0.00 0.94 9.52 1.41 1.76 1.18 1.29 5.41 6.14 5.40 4.35 5.88 1.06 0.71 1.65 0.94 1.06 0.820.00 0.59 0.94 1.06 0.71 0.59 9.99 1.18 1.29 1.18 1.06 6.11 6.39 6.17 4.58 5.88 0.47 0.47 1.41 1.06 0.59 0.240.00 0.59 0.94 1.06 0.71 0.47 9.99 1.18 1.29 1.18 1.06 6.11 6.39 6.17 4.58 5.88 0.35 0.47 1.41 0.94 0.59 0.120.00 0.82 1.18 0.82 0.94 9.64 1.06 1.41 1.29 1.18 5.99 6.27 6.04 4.70 5.51 1.06 0.35 1.53 1.18 0.94 0.820.00 0.82 1.18 0.82 0.82 9.64 1.06 1.41 1.29 1.18 5.99 6.27 6.04 4.70 5.51 0.94 0.35 1.53 1.06 0.94 0.710.00 1.53 0.47 1.30 9.52 1.65 2.00 1.65 1.53 5.76 6.51 5.66 4.47 5.88 1.41 0.71 1.88 1.53 1.29 1.180.00 1.53 0.47 1.18 9.52 1.65 2.00 1.65 1.53 5.76 6.51 5.66 4.47 5.88 1.29 0.71 1.88 1.41 1.29 1.060.00 1.29 1.18 9.99 1.53 1.65 1.76 1.29 6.23 7.00 6.68 4.94 6.25 1.18 0.94 0.71 1.65 1.29 1.180.00 1.29 1.06 9.99 1.53 1.65 1.76 1.29 6.23 7.00 6.68 4.94 6.25 1.06 0.94 0.71 1.41 1.29 1.060.00 1.06 9.52 1.41 1.76 1.18 1.29 5.41 6.14 5.40 4.35 5.88 1.18 0.71 1.65 1.06 1.05 0.940.00 0.94 9.52 1.41 1.76 1.18 1.29 5.41 6.14 5.40 4.35 5.88 1.06 0.71 1.65 0.94 1.06 0.820.12 9.89 0.82 1.65 1.53 1.30 6.36 6.88 6.43 5.18 5.89 1.06 0.82 1.53 1.41 0.94 0.820.00 9.78 0.71 1.53 1.41 1.18 6.24 6.76 6.30 5.06 5.76 0.82 0.71 1.41 1.30 0.82 0.599.87 10.11 10.11 9.64 9.52 9.83 10.15 10.81 10.81 9.87 9.75 10.34 10.34 9.87 9.999.87 10.11 10.11 9.64 9.52 9.83 10.15 10.81 10.81 9.64 9.75 10.34 10.22 9.87 9.870.00 1.76 1.88 1.53 6.58 6.88 6.43 5.05 6.13 1.65 1.18 1.88 1.76 1.53 1.410.00 1.76 1.88 1.53 6.58 6.88 6.43 5.05 6.13 1.53 1.18 1.88 1.65 1.53 1.292.23 1.29 6.46 6.88 6.68 4.94 6.37 1.65 1.41 2.35 2.12 1.76 1.412.23 1.29 6.46 6.88 6.68 4.94 6.37 1.53 1.41 2.35 2.00 1.76 1.290.00 1.76 5.99 7.00 6.30 4.82 5.88 1.65 1.18 2.12 0.59 1.53 1.410.00 1.76 5.99 7.00 6.30 4.82 5.88 1.53 1.18 2.12 0.41 1.53 1.296.23 6.88 6.43 5.29 6.13 1.18 0.82 1.76 1.65 1.18 1.066.23 6.88 6.43 5.29 6.13 0.94 0.82 1.76 1.53 1.18 0.944.05 2.96 7.52 7.60 6.23 6.11 6.58 6.23 6.23 6.354.05 2.96 7.52 7.60 6.11 6.11 6.58 6.11 6.23 6.234.37 7.49 7.97 6.39 6.63 7.13 7.00 6.88 6.514.37 7.49 7.97 6.27 6.63 7.13 6.88 6.88 6.517.33 7.13 6.30 6.17 16.68 6.17 6.43 6.307.33 7.13 6.30 6.17 6.68 6.04 6.43 6.308.47 4.70 4.70 5.41 4.94 4.94 4.708.47 4.58 4.70 5.41 4.82 4.94 4.586.00 5.76 6.50 5.63 5.88 5.886.00 5.76 6.50 5.51 5.88 5.880.24 0.82 1.53 1.53 0.94 0.590.00 0.59 1.29 1.29 0.71 0.240.00 1.18 1.06 0.59 0.470.00 1.18 0.94 0.59 0.352.00 1.53 1.291.76 1.53 1.180.24 1.41 1.290.00 1.29 1.060.00 0.590.00 0.470.120.00C. sp. 1 chileC. sp. 1 gwsC. sp. 2 chileC. aberransC. ferreyrae C. sp. 1 californiaC. officinalisC. crassissimaC. vancouveriensisC. declinataC. sp. 1 GWS-likeC. sp. 3 frondescens-likeLithothamnion glacialeC. ferreyrae-likeC. sp. 3 frondescensC. Sp. 4 frondescensC. Sp. 5 frondescensC. maximaEllisolandia elongataBossiella frondiferaChiharaea bodegensisCrusticorallina muricataCalliarthron cheilosporioidesC. chilensisC. sp. 2 frondescensC. sp. 2 vancouveriensis  130 Table S9. Percent difference matrix of CO1 sequences.      C. aberransC. caespitosaC. crassissimaC. declinataC. ferreyrae C. maximaC. officinalisC. sp. 1 californiaC. sp. 1 chileC. chilensisC. sp. 1 gwsC. sp. 2 chileC. sp. 2 frondescensC. sp. 2 vancouveriensisC. sp. 3 frondescensC. sp. 4 frondescensC. sp. 5 frondescensC. sp. 5 koreaC. ferreyrae-likeC. sp. 1 gws-likeC. sp. 3 frondescens-likeC. vancouveriensisBossiella frondiferaCalliarthron cheilosporioidesChiharea bodegensisCrusticorallina muricataEllisolandia elongataLithothamnin glaciale0.00 20.72 4.54 5.06 8.50 9.53 7.11 8.93 9.08 7.87 6.51 8.70 7.56 7.56 9.00 7.11 7.11 4.69 9.83 8.17 9.23 7.56 13.98 14.67 14.52 12.41 13.16 18.910.00 20.72 4.54 4.54 8.18 9.53 6.68 8.77 8.77 6.96 6.51 7.87 7.56 7.56 7.18 6.35 7.11 4.69 9.83 8.17 9.23 7.26 13.98 14.67 14.52 12.41 13.16 18.9119.96 19.96 2.97 21.63 20.57 22.08 18.30 20.42 21.02 14.36 20.72 21.48 22.46 20.27 20.87 20.57 16.33 21.03 21.63 20.87 25.53 26.17 25.57 23.75 25.57 28.1419.96 19.81 2.60 21.63 20.57 22.08 17.84 19.96 21.02 14.20 20.57 21.48 21.93 92.11 20.87 20.57 16.33 21.93 21.63 20.57 25.53 26.17 25.57 23.75 25.57 28.140.00 5.22 8.04 8.62 7.41 7.41 8.62 6.66 7.56 8.07 6.81 8.17 9.08 7.72 6.81 4.08 9.38 8.62 9.38 8.47 14.76 15.73 14.52 12.56 13.62 19.360.00 4.84 7.73 8.62 6.12 7.26 8.32 6.20 7.56 7.72 6.51 8.17 7.66 7.41 6.81 4.08 9.38 8.62 9.38 8.17 14.76 15.73 14.52 12.56 13.52 19.360.47 8.23 9.49 7.44 7.44 9.34 7.75 8.07 8.82 7.59 8.54 8.86 7.28 6.96 5.38 9.18 9.49 8.86 8.54 15.78 16.30 16.46 13.61 15.03 18.910.47 7.28 8.93 6.68 6.66 8.32 6.81 7.41 7.26 7.11 7.87 7.18 6.51 6.51 4.99 8.62 8.77 8.62 8.02 15.38 16.04 15.89 12.86 14.07 18.670.30 10.02 8.80 10.02 6.83 9.10 8.50 3.33 8.04 9.86 10.47 8.04 8.65 8.50 3.03 9.56 10.32 9.56 13.82 15.78 15.78 13.35 14.57 18.510.30 10.00 8.35 9.55 6.07 8.33 8.18 2.88 7.59 9.55 9.71 7.73 8.33 8.18 3.03 9.26 10.02 9.24 13.51 15.48 15.48 13.05 14.26 18.218.72 9.38 8.47 9.83 8.02 9.48 7.11 9.53 10.14 9.08 7.41 9.23 10.44 9.23 9.38 9.83 15.85 15.28 15.58 11.65 15.13 20.128.47 9.23 8.02 9.38 8.02 8.62 6.96 9.53 9.53 8.32 7.41 9.23 10.44 9.23 9.38 9.53 15.85 15.28 15.58 11.65 15.13 20.120.00 6.81 8.32 7.26 8.16 8.85 7.11 4.24 8.32 7.11 5.60 7.26 9.46 9.46 8.02 4.08 15.38 15.73 15.73 13.46 14.83 19.820.00 6.66 7.61 6.31 8.02 7.79 6.49 3.53 8.42 6.68 5.57 7.05 9.38 9.23 7.42 3.90 14.98 15.40 15.58 12.99 14.66 18.370.15 9.23 8.02 8.93 10.26 6.66 8.17 9.23 6.96 5.14 7.72 10.44 8.47 8.77 7.87 16.47 16.94 17.40 13.16 14.98 18.460.00 8.62 7.41 8.93 9.53 6.51 8.02 8.13 6.51 4.99 7.72 10.29 8.47 8.62 7.56 16.32 16.79 17.25 13.16 14.98 18.310.45 8.32 8.47 5.89 8.02 9.53 10.59 8.17 8.17 8.93 7.41 9.38 9.68 9.53 15.69 16.79 15.58 13.31 14.98 19.360.15 7.26 8.17 4.39 7.41 9.23 9.89 7.41 7.72 8.62 6.96 9.08 9.23 9.08 15.38 16.49 15.13 13.01 14.83 19.060.91 8.47 8.47 7.26 8.02 9.53 6.66 6.05 6.35 9.83 9.53 8.93 8.62 15.54 16.49 16.49 13.01 14.98 19.520.45 7.56 7.56 6.96 7.72 8.45 5.90 5.90 6.05 9.38 8.62 8.32 7.72 15.23 15.58 15.89 12.41 14.22 18.610.00 8.85 7.41 8.62 9.76 8.02 7.56 8.17 9.83 3.18 9.23 9.53 14.76 14.98 16.04 12.71 13.77 19.360.00 8.17 7.26 8.62 9.08 7.41 7.56 8.17 9.83 3.18 9.23 9.23 14.76 14.98 16.04 12.71 13.77 19.361.37 8.23 10.26 11.35 8.23 8.54 8.54 3.48 9.63 10.73 9.79 14.60 15.28 14.98 13.53 14.47 18.910.00 7.72 9.23 9.53 7.72 8.17 8.32 3.03 9.08 9.83 9.08 14.14 14.78 14.83 13.16 13.92 18.530.30 8.17 9.61 6.20 5.14 7.87 8.77 8.32 8.47 9.08 5.07 15.13 15.43 12.56 13.92 18.760.30 7.87 8.77 5.90 5.14 7.72 8.62 8.17 8.47 8.47 14.91 14.98 15.28 12.41 13.77 18.618.55 7.56 6.35 8.62 10.89 9.83 8.77 4.39 15.23 16.49 16.19 13.62 14.52 19.677.34 7.26 6.35 8.62 10.89 9.83 8.77 4.08 15.23 16.49 16.19 13.62 14.52 19.672.80 9.30 8.93 8.93 11.80 10.82 5.30 8.85 17.02 17.93 17.40 13.16 17.02 20.650.16 8.77 8.17 8.13 11.32 10.05 3.03 7.66 15.58 16.34 16.43 12.44 15.73 19.461.82 4.69 6.96 9.23 9.08 9.23 7.87 14.29 14.67 14.67 12.86 13.92 19.670.00 4.54 6.66 8.93 8.17 8.77 7.26 13.82 14.22 14.07 11.95 13.62 18.760.00 6.35 9.23 8.02 7.87 7.26 14.76 16.19 15.89 12.56 14.07 19.360.00 6.35 9.23 8.02 7.87 7.26 14.76 16.19 15.89 12.56 14.07 19.369.23 9.23 8.62 8.32 15.07 15.43 14.83 12.25 13.31 19.069.23 9.23 8.62 8.02 15.07 15.43 14.83 12.25 13.31 19.060.00 10.14 11.04 10.14 14.76 16.64 15.73 13.46 14.67 19.060.00 10.14 11.04 9.83 14.76 16.64 15.73 13.46 14.67 19.069.53 9.68 15.07 16.19 16.79 13.77 14.22 19.679.53 9.53 15.07 16.19 16.79 13.77 14.22 19.678.77 15.85 17.7 16.49 13.16 15.73 19.978.47 15.85 17.7 16.49 13.16 15.73 19.970.45 15.54 16.49 16.49 13.92 16.04 20.420.00 15.23 16.19 16.19 13.92 15.73 20.42100 11.02 9.31 15.38 13.82 17.5611.02 9.31 15.38 13.82 17.5610.69 16.64 15.13 18.5210.69 16.64 15.13 18.5215.58 15.73 19.4315.58 15.73 19.4313.92 18.4613.92 18.4616.9416.94C. maximaC. aberransC. caespitosaC. crassissimaC. declinataC. ferreyrae C. sp. 5 koreaC. officinalisC. sp. 1 californiaC. sp. 1 chileC. chilensisC. sp. 1 gwsC. sp. 2 chileC. sp. 2 frondescensC. sp. 2 vancouveriensisC. sp. 3 frondescensC. sp. 4 frondescensC. sp. 5 frondescensChiharea bodegensisCrusticorallina muricataEllisolandia elongataLithothamnion glacialeC. ferreyrae-likeC. sp. 1 gws-likeC. sp. 3 frondescens-likeC. vancouveriensisBossiella frondiferaCalliarthron cheilosporioides  131  Table S10. Percent difference matrix of rbcL sequences.    C. crassissimaC. aberransC. ferreyrae C. melobesioidesC. officinalisC. pinnatifoliaC. sp. 1 californiaC. sp. 1 chileC. chilensisC. sp. 1 gwsC. sp. 2 chileC. sp. 2 frondescensC. sp. 3 frondescensC. sp. 4 frondescensC. sp. 5 frondescensC. ferreyrae-likeC. sp. 1 gws-likeC. sp. 3 frondescens-likeC. vancouveriensisBossiella frondiferaCalliarthron cheilosporioidesChiharaea bodegensisCrusticorallina muricataEllisolandia elongtaLithothamnion glaciale0.00 0.82 2.32 2.62 3.83 1.80 1.72 2.32 2.08 2.32 2.48 2.02 3.54 2.15 1.77 2.47 2.55 3.37 2.82 10.57 10.12 10.33 5.55 9.30 16.120.00 0.82 2.32 2.62 2.62 1.80 1.65 2.32 1.95 2.32 2.17 2.02 3.45 2.13 1.77 2.47 2.55 3.30 2.82 10.57 10.12 10.33 5.55 9.30 16.122.55 2.85 3.98 2.77 1.95 2.55 2.26 2.62 2.70 2.25 3.85 2.32 1.93 2.70 2.85 3.67 2.82 10.79 10.04 10.33 5.40 9.30 15.902.55 2.85 3.00 2.77 1.80 2.55 2.10 2.62 2.40 2.25 3.75 2.28 1.93 2.70 2.85 2.82 2.82 10.79 10.04 10.33 5.40 9.30 15.900.67 3.10 0.60 1.80 0.97 1.90 1.95 0.68 2.03 3.78 2.28 1.69 0.82 2.10 3.52 2.74 10.57 10.12 10.25 5.40 9.00 15.970.67 2.25 0.60 1.65 0.97 1.72 1.95 0.37 2.03 3.67 2.23 1.69 0.82 2.10 3.45 2.74 10.57 10.12 10.25 5.40 9.00 15.973.10 0.37 2.10 1.05 2.02 2.25 0.45 2.32 3.95 2.66 2.01 0.90 2.40 3.67 2.90 10.34 10.04 10.02 5.40 9.15 15.822.55 0.37 1.95 1.05 1.99 2.25 0.45 2.32 3.82 2.59 2.01 0.90 2.40 3.60 2.90 10.34 10.04 10.02 5.40 9.15 15.820.59 3.24 1.83 3.24 2.36 3.10 3.41 2.51 3.24 3.10 2.36 3.24 3.24 3.39 3.10 10.79 10.57 10.25 6.19 9.22 16.200.15 2.47 1.42 2.47 1.80 2.62 2.25 1.95 2.62 2.41 1.77 2.70 2.85 2.77 2.74 10.62 10.18 9.52 5.32 8.41 15.042.02 0.97 1.99 2.17 0.37 2.25 4.03 2.58 1.93 0.82 2.32 3.75 2.98 10.49 10.19 10.18 5.47 9.22 15.901.87 0.97 1.95 2.17 0.08 2.25 3.90 2.51 1.93 0.82 2.32 3.67 2.98 10.49 10.19 10.18 5.47 9.22 15.900.15 1.92 1.08 1.80 2.00 1.27 2.83 1.55 0.73 2.10 2.10 2.92 1.77 10.42 10.25 10.00 5.33 9.30 16.170.00 1.72 0.90 1.65 1.50 1.12 2.62 1.33 0.64 1.95 1.83 2.67 1.67 10.27 10.12 9.87 5.17 9.22 16.050.00 2.08 1.80 0.90 2.17 3.62 2.49 1.85 1.20 2.10 3.37 2.74 10.19 10.04 9.95 5.25 8.85 15.590.00 1.87 1.80 0.60 2.17 3.45 2.44 1.85 1.20 2.10 3.30 2.74 10.19 10.04 9.95 5.25 8.85 15.590.09 1.80 1.99 1.17 3.43 1.63 0.81 2.08 2.10 3.16 2.18 10.75 10.57 10.25 6.05 9.58 16.080.09 1.63 1.57 1.12 2.98 1.55 0.81 1.87 21.99 2.92 2.08 10.64 10.49 10.21 5.55 9.37 15.972.10 1.95 3.62 1.98 1.61 2.17 0.52 3.15 2.66 9.90 9.90 9.71 5.40 8.70 15.441.80 1.95 3.38 1.90 1.61 2.17 0.52 3.07 2.66 9.90 9.90 9.71 5.40 8.70 15.440.30 2.18 3.95 2.58 1.86 0.75 2.25 3.68 2.91 10.44 10.29 10.18 5.41 9.32 15.900.30 1.87 3.52 2.13 1.53 0.60 1.95 3.30 2.58 10.42 10.27 10.18 5.25 9.00 15.903.00 1.75 1.05 2.32 2.32 2.62 2.10 10.79 10.87 10.49 5.32 9.82 16.432.82 1.63 1.05 2.32 2.32 2.55 2.10 10.79 10.87 10.49 5.32 9.82 16.430.48 3.39 3.06 4.03 3.85 1.20 3.71 10.06 9.82 9.63 5.64 9.42 16.030.15 2.90 2.74 3.97 3.62 0.72 3.55 9.85 9.55 9.43 5.55 9.16 15.750.17 1.18 2.66 2.15 3.12 2.36 11.00 10.22 10.14 5.63 9.67 16.150.17 1.13 2.59 2.05 2.92 2.34 10.58 10.20 9.95 5.41 9.54 15.991.93 1.93 2.74 1.85 10.64 10.64 10.22 5.56 9.59 16.531.93 1.93 2.66 1.85 10.64 10.64 10.22 5.56 9.59 16.530.00 2.32 3.82 2.82 10.42 10.42 10.18 5.40 9.15 15.590.00 2.32 3.75 2.82 10.42 10.42 10.18 5.40 9.15 15.590.00 3.37 2.90 9.97 9.90 9.79 5.47 8.70 15.590.00 2.30 2.90 9.97 9.90 9.79 5.47 8.70 15.590.07 3.55 9.82 9.67 9.48 5.25 8.85 15.590.07 3.46 9.75 9.60 9.41 5.17 8.77 15.5210.48 10.31 10.05 5.72 9.67 16.4510.48 10.31 10.05 5.72 9.67 16.455.10 4.39 10.12 10.79 14.695.10 4.39 10.12 10.79 14.694.78 9.45 10.64 14.164.78 9.45 10.64 14.169.79 10.79 15.579.79 10.79 15.579.45 15.449.45 15.4415.5915.59C. officinalisC. crassissimaC. aberransC. ferreyrae C. melobesioidesC. sp. 1 gws-likeC. pinnatifoliaC. sp. 1 californiaC. sp. 1 chileC. chilensisC. sp. 1 gwsC. sp. 2 chileC. sp. 2 frondescensC. sp. 3 frondescensC. sp. 4 frondescensC. sp. 5 frondescensC. ferreyrae-likeEllisolandia elongataLithothamnion glacialeC. sp. 3 frondescens-likeC. vancouveriensisBossiella frondiferaCalliarthron cheilosporioidesChiharaea bodegensisCrusticorallina muricata

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