Creation of a Species-Level Cephalopod Phylogeny to Resolve Major and Minor Clades Sriram V. Ramamurthy¹, Bridget A. Vincent², Emily S. Lau², Todd H. Oakley² 1 University of California Santa Barbara, College of Creative Studies 2 University of California Santa Barbara, Dept. of Ecology, Evolution, and Marine Biology Introduction Phylogenetic Analysis

• Cephalopods (Mollusca : Cephalopoda) are a diverse Class of Sanchez et al. 2018 Originally Genus-Level Lindgren et al. 2012 Originally Species-Level Our Phylogeny Originally Species-Level marine invertebrates consisting of over 800 described species.

• The systematic relationships of and between many clades of Error cephalopods are still unclear, demonstrating the need for a robust Clade A phylogeny. Major Cephalopod Lineages

Subclass Nautiloidea Includes the pelagic Nautilidae; presence of an elaborate aragonite shell. An ancient lineage.

Octopodiformes: Eight arms. Typically includes groups such as the cirrate octopuses (internal

shell, two fins on head, and cirri on Error tentacles present) and incirrate octopuses (absence of these S. Chantra features). Subclass Coleoidea Decapodiformes: Eight tentacles and two arms. Typically includes groups such as the squids (Presence of a internal chitinous structure called the "gladius"), cuttlefishes (Presence of an internal calcified structure called the "sepion") and bobtail squids Nick Hobgood (reduced or absent gladius, absence of sepion).

• Previous studies' phylogenies are often poorly resolved, meaning that the relationships shown in the phylogeny are not highly probabilistic. In addition, poor taxon sampling and www.vox.com inadequate sampling strategies, such as picking one species to represent a genus, lead to inaccurate groupings.

• Resolving the evolutionary relationships within a group forms the basis for studying ecological strategies that influence its diversification, complex trait evolution, and predicting how species will respond to anthropogenic stressors.

Bioluminescence: A Complex Trait

Complex traits functionally Symbiotic Autogenic integrate many genes while bioluminescence: Light is bioluminescence: Light simple traits use a few genes. Bioluminescence, the produced by symbiotic bacteria is produced by the organism biological production and living within the photophore. itself, through a chemical emission of light, involves reaction. complex morphological and physiological specialization. Photophores are specialized organs containing Autogenic bioluminescence-producing Photophores * Family-level phylogenies are shown for the sake of clarity. with structures, molecules, or Symbiotic Luminescent Photophores symbionts. Many Chemicals cephalopods are with Bacteria Results Discussion and Future Directions bioluminescent, which they Vibrio fischeri use for defense, courtship, Coelentrazine + • We found that while aspects of our phylogeny agree with some previous studies, the prey-capture, and more. Luciferase Sanchez et al. 2018 Lindgren et al. 2012 Our Phylogeny relationships within Cephalopoda become complicated when analyzed at the species level. • Two major Coleoid lineages • Two major Coleoid lineages • Two major Coleoid lineages Objectives • Oegopsida (All squid families • Oegopsida (All squid families • Oegopsida (All squid families • There is some agreement with previous studies, such as the recovery of two major except Loliginidae) is paraphyletic except Loliginidae) is monophyletic except Loliginidae) is paraphyletic, Coleoid lineages and the monophyly of the cirrate octopuses. • Resolve problematic relationships within Cephalopoda by creating (internal placement of Sepiolidae internal placement of Myopsida and Sepiadariidae) (Loliginidae) and Sepiida • Several major clades such as Oegoposida, Myopsida, and Octopodidae are not a species-level phylogeny to assess major and minor clades. monophyletic and are in need of further taxonomic revision. • Myopsida (Loliginidae) is • Myopsida (Loliginidae) is • Myopsida (Loliginidae) is • Compare the placement of major and minor clades in our monophyletic monophyletic polyphyletic • We show different placements of other minor clades such as Spirulidae, phylogeny to other published phylogenies. • Vampyroteuthidae within • Vampyroteuthidae sister to cirrate • Vampyroteuthidae within Vampyroteuthidae, and Gonatidae. The placement of the Vampyroteuthidae within the Decapodiformes disagrees with its placement in the two other studies analyzed. • Use this phylogeny as a tool to form and test phylogenetic Octapodiformes and sister to all and incirrate octopuses Decapodiformes and sister to octopuses Pyroteuthidae hypotheses. • We are using this phylogenetic framework to conduct ancestral state reconstruction • Cirrate octopuses • Cirrate octopuses • Cirrate octopuses of bioluminescence to determine the number of times this trait originated within Methods for Phylogenetic Reconstruction (Opisthoteuthidae, Cirroctopodidae, (Opisthoteuthidae, Cirroctopodidae, (Opisthoteuthidae, Cirroctopodidae, Cephalopoda. Publicly available short-read genetic data from NCBI was used, Cirroteuthidae, Stauroteuthidae) are Cirroteuthidae, Stauroteuthidae) are Cirroteuthidae, Stauroteuthidae) are with the following molecular markers being used: 12S, 16S, paraphyletic monophyletic monophyletic • We can use this phylogeny to study the predictability and repeatability of evolution 28S, actin, COI, COIII, Cyt-b, histone, myosin, ODH (octopine • Octopodidae is monophyletic • Octopodidae is paraphyletic • Octopodidae is polyphyletic and the evolutionary dynamics of complex traits. dehydrogenase), Pax-6, rhodopsin, 18S spacer region. The Relevant literature: following programs were used: MAFFT, a multiple sequence • Gonatidae is monophyletic • Gonatidae is monophyletic • Gonatidae is polyphyletic 1. Lindgren, A. R., Pankey, M. S., Hochberg, F. G., & Oakley, T. H. (2012). A multi-gene phylogeny of Cephalopoda supports convergent morphological evolution in alignment program, was used to create gene family alignments; association with multiple habitat shifts in the marine environment. BMC evolutionary biology, 12(1), 129. • Spirulidae forms a monophyletic • Spirulidae is monophyletic with • Spirulidae forms a paraphyletic 2. Sanchez, G., Setiamarga, D. H., Tuanapaya, S., Tongtherm, K., Winkelmann, I. E., Schmidbaur, H., ... & Gleadall, I. (2018). Genus-level phylogeny of cephalopods IQ-TREE to create gene trees, and ASTRAL to concatenate the using molecular markers: current status and problematic areas. PeerJ, 6, e4331. group with Idiosepiidae, Loliginidae, Oegopsid and Myopsid squids group with Idiosepiidae, Loliginidae, Acknowledgements: I would like to extend a sincere thank you to Bridget Vincent and Emily Lau for their mentorship, and to Dr. Todd gene trees into one species tree. Oakley for his wholehearted support. Use was made of computational facilities purchased with funds from the National Science Foundation and Sepiidae and Sepiidae (CNS-1725797) and administered by the Center for Scientific Computing (CSC). The CSC is supported by the California NanoSystems Institute and the Materials Research Science and Engineering Center (MRSEC; NSF DMR 1720256) at UC Santa Barbara.