Development, Anatomy, and Phylogenetic Relationships of Jawless Vertebrates and Tests of Hypotheses about Early Vertebrate Evolution by Tetsuto Miyashita A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Systematics and Evolution Department of Biological Sciences University of Alberta © Tetsuto Miyashita, 2018 ii ABSTRACT The origin and early evolution of vertebrates remain one of the central questions of comparative biology. This clade, which features a breathtaking diversity of complex forms, has generated profound, unresolved questions, including: How are major lineages of vertebrates related to one another? What suite of characters existed in the last common ancestor of all living vertebrates? Does information from seemingly ‘primitive’ groups — jawless vertebrates, cartilaginous fishes, or even invertebrate outgroups — inform us about evolutionary transitions to novel morphologies like the neural crest or jaw? Alfred Romer once likened a search for the elusive vertebrate archetype to a study of the Apocalypse: “That way leads to madness.” I attempt to address these questions using extinct and extant cyclostomes (hagfish, lampreys, and their kin). As the sole living lineage of jawless vertebrates, cyclostomes diverged during the earliest phases of vertebrate evolution. However, precise relationships and evolutionary scenarios remain highly controversial, due to their poor fossil record and specialized morphology. Through a comparative analysis of embryos, I identified significant developmental similarities and differences between hagfish and lampreys, and delineated specific problems to be explored. I attacked the first problem — whether cyclostomes form a clade or represent a grade — in a description and phylogenetic analyses of a new, nearly complete fossil hagfish from the Cenomanian of Lebanon. Aided by a detailed analysis of morphological characters, new phylogenetic trees recovered cyclostomes as a clade. This is the first morphological phylogeny to yield cyclostomes as monophyletic and therefore helps reconcile a major point of conflict between morphology- and molecule-based phylogenetics. iii I tested the second problem — the assumption that living lampreys pass through a filter- feeding larval stage resembling ancestors of vertebrates — using a growth series of a fossil lamprey from the Late Devonian of South Africa. Surprisingly, these fossil lamprey larvae bear little morphological resemblance to larvae of living lampreys. Instead, the growth series reveals ontogenetic transition of traits that are consistent with the predatory habit of modern lamprey adults. Through comparison to other Paleozoic stem lampreys, I suggest that the filter-feeding larval stage evolved independently within vertebrates. Under this new scenario, larvae of living lampreys are a poor model with which to reconstruct primitive vertebrate characters. For the third problem — how biting jaws evolved in vertebrates — I focused on one key feature of the jaw apparatus: the jaw joint. Using gene expression profiles and gene knockouts in lamprey and zebrafish embryos, I tested three hypotheses proposed to explain the origin of the jaw joint. Preliminary results suggest that a jaw joint may originate from a blood sinus pinched between two cartilages, or from a type of immature cartilaginous tissue. I describe a new genetic line of zebrafish that carries a mutation in the homeodomain-coding sequence of nkx3.2 — a jaw joint marker gene. Homozygous mutants have fused upper and lower jaw cartilages, replicating nkx3.2 morphants. At the adult stage, the mutants developed craniofacial phenotypes that resemble some of the jawless stem gnathostomes. Finally, I propose an analytical pipeline to address a number of remaining questions in early vertebrate phylogeny: supertrees as a platform for future modular analyses of morphological characters. By sorting individual characters by different biological attributes, I hope to illustrate a view on early vertebrate evolution through understanding the dynamics of character evolution. iv PREFACE This thesis is an original work by Tetsuto Miyashita. The research, of this thesis is a part, received research ethics approval from the University of Alberta Research Ethics Board, title “Evolutionary origin of vertebrate jaws” on May 4, 2014, under ID AUP00000793. Some of the research conducted for this thesis forms part of collaborative projects: Chapter 1 of this thesis is a revised version of a chapter for a book in press (Evolution and Development of Fishes, Cambridge University Press; edited by Z. Johanson, M. Richter, and C. Underwood). Title: Comparative development of cyclostomes. Authors: Tetsuto Miyashita, Stephen A. Green, Marianne E. Bronner. The manuscript was drafted by me. S.A.G. contributed photographs for Fig. 1.4. M.E.B. provided resources with which to conduct this work at California Institute of Technology. Chapter 2 of this thesis presents results from collaboration led by Philip Currie at the University of Alberta. Synchrotron radiation scanning was conducted and the data processed by Phillip Manning at Mace Brown Museum of Natural History and colleagues. All other results, including interpretation of the results from synchrotron radiation scanning, are my own. Chapter 3 of this thesis presents results from collaboration with Robert Gess at Albany Museum, who collected specimens described in the chapter. All results, including illustrations, phylogenetic and morphological analyses, and anatomical interpretations, are my own, unless cited otherwise. Chapter 4 of this thesis presents results from collaboration with Ted Allison at the University of Alberta and Marianne Bronner at California Institute of Technology. Both Allison and Bronner provided laboratory space, resources, and animals. All results and interpretations are my own, unless cited otherwise. v !"#$"%&'" ( )"*+'&",&'" -.+ /" &"')0'1($' ,"2'3"+4, +.+ ( -. $.2.5 62.&"-., &. +'-'%'7 '& ')(-."-. The infinitely unfathomable is as essential for man as the little planet on which he dwells. — Demons, Fyodor Dostoevsky セロニアス・モンクはあの不可思議な和音を、理屈や論理で考え出したわけじゃあらない。彼はただしっ かり目を見開いて、それを意識の暗闇の中から両手ですくい上げただけなのだ。大事なのは無から何かを 創りあげることではあらない。諸君のやるべきはむしろ、今そこにあるものの中から、正しいものを見つ け出すことなのだ。 Thelonius Monk didn’t need theories or logics to come up with those inexplicable chords. He only stared into darkness, keeping his eyes peeled and reaching out for the unfathomable. Nobody is asking y’all to create something out of nothing. Finding something right from what y’all have already got there — that’s precisely what y’all gotta do. — Killing Commendatore, Haruki Murakami (Translation by T.M.) vi ACKNOWLEDGEMENTS When I entered the graduate program, my advisor Rich Palmer gave me Steven Weinberg’s commencement address “Four Golden Lessons”. In retrospect, the golden lessons were a development plan for me. As true as those lessons were, however, Weinberg overlooked one crucial item: the value of trust. The freedom and support given to me was the gift of confidence he had in me that — perhaps not so earned at the time but eventually — I will put it to a good use. I will continue to feel that confidence as I move on. Thank you, Rich. I will be measuring my success by whether you would be proud of (not so much what I accomplish but) how I develop myself as a student of biology. Using baseball reference, you have been a great manager. You took some heat for me, and still sent me out there to pitch a game. If Rich was my equivalent of an MLB manager, Philip Currie would be that coach who believed in me through the farm system. No one else watched me growing up as long as you did, or brought me all the way up from the rookie league. Phil, I will be looking back to you constantly for exactly the same reasons with Rich. Keeping on this metaphor, Eva Koppelhus was my great General Manager without whom I would not have a contract with this team. I have had an unparalleled squad of amazing coaches — Michael Coates, Ted Allison, Marianne Bronner, Philippe Janvier, Richard Strathmann, Richard Behringer, and Brian Hall. They served as ad hoc advisors, provided their lab spaces and resources for my thesis research, and took me under their wing when I was playing a visitor game. Of course, I would not be able to play without the ground-keeping crew, physical training team, and clubhouse staff — they are technicians in the labs who facilitated my work, collection managers and assistants at the museums who provided access to the specimens, staff at marine labs who let me have all the fun in the world (but not more than all that is there), field assistants who protected me from killing myself doing something stupid, library staff who went on a mission to obtain obscure references, department staff who made sure I am signing my real name in the right spot, and the University of Alberta IBD Clinic that has been providing my lifeline since 2009. They are too numerous to name individually here, but they will know when they find a box of chocolates. vii It has been an honour to take the field with a talented and inspired group of players. First and foremost, they include my course mates from the 2010 Larval Biology class at Friday Harbor Laboratories and the 2013 Embryology class at the Marine Biological Laboratory. No words can describe the immense pleasure I felt to spend days, nights, and all hours in-between with people who share values and philosophies about science. It seemed as though I had lived just to share in these short but