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Molecular contributions to species descriptions of dicyemid mesozoans Eshragh, Roya Marie 2013

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	 ?MOLECULAR CONTRIBUTIONS TO SPECIES DESCRIPTIONS OF DICYEMID MESOZOANS   by   Roya Marie Eshragh  B.S., Michigan State University, 2009     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE   in   The Faculty of Graduate and Postdoctoral Studies  (Zoology)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    October 2013     ? Roya Marie Eshragh, 2013   	 ? ii	 ?Abstract  Dicyemids are enigmatic parasites found only within the excretory systems of benthic cephalopods. Over the past century, dicyemids have been considered to be either complex protozoa, ?mesozoa? that are ambiguously intermediate between protozoa and metazoa, or reduced metazoans. The phylogenetic position and overall diversity of dicyemids is poorly understood. Current species identification criteria are unconvincing because they are based solely on morphological traits. I set out to test current morphological species concepts with DNA barcodes from dicyemids collected from Pacific Northwest cephalopods. Variation within sequences of the small subunit (18S) rRNA gene was explored because this marker (1) is known to be fast-evolving in parasitic eukaryotes, (2) is one of the few molecular markers to have been previously sequenced in some dicyemids, and (3) has been used successfully as a barcode in other groups of parasites. Three host species of cephalopods were collected in this study, each hosting multiple historical morphospecies of dicyemid parasites. Thirty-four individual dicyemids encompassing eight morphospecies were isolated and their 18S rDNA sequenced. Molecular phylogenetic analyses of these data were incongruent with current morphology-based species concepts. The 18S rDNA sequences suggest that each host species of cephalopod harbors only one species of dicyemid with a great deal of morphological variation. However, the 18S rDNA sequences should eventually be tested with other rapidly evolving molecular markers. Attempts were made to sequence the mitochondrial cytochrome oxidase I (COI) gene, the mitochondrial 16S rRNA gene, and both Internal Transcribed Spacers (ITS) of the nuclear rRNA operon. With so little of the dicyemid genome known, I was unable to establish reliable primer pairs for these genes within the time constraints of my MSc thesis. Nonetheless, this study has shown that DNA barcoding is a powerful tool for the delimitation of dicyemid species. Understanding the diversity of parasite species is particularly problematic because they tend to be devoid of consistent (informative) morphological traits while simultaneously rich in morphological variation associated with developmental stages and environmental conditions.  The addition of DNA barcodes to dicyemid diversity will simplify and improve species boundaries in a lineage that is difficult to define in every aspect.             	 ? iii	 ?Preface   This project was identified, designed and executed by myself with inspiration from Brian Leander and his entire lab. As I was the first in the lab to look at dicyemids, the field was relatively open for me to choose a direction to explore. My idea to collaborate with BC Spot Prawns was inspired by Stephanie Avery-Gomm. Once a viable plan for obtaining local cephalopods was in place, I was able to devise a strategy for obtaining dicyemids and their molecular data. With guidance from Kevin Wakeman, Naoji Yubuki, Thierry Hedger and Sarah Sparmann, I designed all primer pairs and completed all PCR and sequencing steps. I extracted all the data and was able to analyze the results with Sarah?s help.  The whole lab supported me throughout the process and was around to give guidance when questions arose. Brian was always willing to point me in the correct direction and help to find answers when I was stuck. Publications are in progress, but none has been completed yet.             	 ? iv	 ?Table of Contents Abstract ............................................................................................................................. ii Preface .............................................................................................................................. iii Table of Contents .............................................................................................................. iv List of Tables ...................................................................................................................... v List of Figures ................................................................................................................... vi Acknowledgements ........................................................................................................... vi Dedication ....................................................................................................................... viii Chapter 1 ............................................................................................................................ 1 1.1 Introduction .......................................................................................................................... 1 1.1.1. Parasitism of cephalopods ............................................................................................. 1 1.1.2. Dicyemid-host interactions ........................................................................................... 2 1.1.3. The life cycle and morphology of dicyemids ................................................................ 3 1.1.4. The uncertain molecular phylogenetic position of dicyemids ....................................... 5 1.1.5. Dicyemid systematics .................................................................................................... 7 1.1.6. DNA barcoding ............................................................................................................. 8 1.1.7. Using molecular data to validate dicyemid species ..................................................... 10 Chapter 1 Figures .................................................................................................................... 11 Chapter 2 .......................................................................................................................... 15 2.1. Synopsis ............................................................................................................................. 15 2.2. Methods ............................................................................................................................. 16 2.2.1. Collection of specimens and light microscopy ........................................................... 16 2.2.2. Host DNA extraction, PCR, cloning, and sequencing ................................................ 17 2.2.3. Dicyemid DNA extraction, PCR, cloning, and sequencing ........................................ 18 2.2.4. Sequence alignments and molecular phylogenetic analyses: ...................................... 19 2.2.5. Scanning electron microscopy: ................................................................................... 19 2.3 Results ................................................................................................................................. 20 2.3.1 Dicyemid species collected .......................................................................................... 20 2.3.2 Molecular phylogeny analyses ..................................................................................... 20 2.3.3 Scanning electron microscopy ..................................................................................... 21 2.4 Discussion: .......................................................................................................................... 22 2.4.1 Phylogeny ..................................................................................................................... 22 2.4.2 Specificity and Coevolution ......................................................................................... 24 2.4.3 SEM images ................................................................................................................. 27 Chapter 2 Figures .................................................................................................................... 28 Chapter 2 Tables ...................................................................................................................... 31 Chapter 3 .......................................................................................................................... 32 3.1 Conclusion: ......................................................................................................................... 32 3.1.1 Status of species ........................................................................................................... 32 3.1.2 The strengths and weaknesses of molecular phylogenetic data ................................... 33 3.1.3. Dicyemid diversity in the age of molecular biology ................................................... 35 Works Cited ..................................................................................................................... 39  	 ? v	 ?List of Tables Chapter 2 Tables ...................................................................................................................... 32 Table 2.1 ? Dicyemids and Hosts collected .......................................................................... 32                                       	 ? vi	 ?List of Figures  Chapter 1 Figures .................................................................................................................... 12 Figure 1.1 ? Vermiform Dicyemid ........................................................................................ 12 Figure 1.2 ? Known Life Stages in the Dicyemid Lifecycle ................................................. 13 Figure 1.3 ? Dicyemid Infusiform Larva .............................................................................. 14 Figure 1.4 ? Relationship Between the 18S Sequences of Dicyemids and Other Eukaryotes ............................................................................................................................................... 14 Figure 1.5 ? Pictorial Description of Dicyemid Genera ........................................................ 15 Figure 1.6 ? Assumed Phylogenetic Relationship of Dicyemidae Genera ........................... 15  Chapter 2 Figures .................................................................................................................... 29 Figure 2.1 ? Pictorial Descriptions of Local Dicyemid Morphospecies ............................... 29 Figure 2.2 ? Local Dicyemid Isolates ................................................................................... 30 Figure 2.3 ? ML Tree of All Current Dicyemid 18S Sequences ........................................... 31               	 ? vii	 ?Acknowledgements None of this work could have been accomplished without the support and guidance of my advisor, committee, friends and coworkers. Additional thanks to Dr. Brian Leander for taking me as his student under special circumstances and giving me such a valuable opportunity.  Thank you to BC Spot Prawns for contributing the most important part of this journey ? the host species! Thank you a thousand times for your generosity and the wonderful experience it led to. Every part of this thesis was dependent on the ability to collect cephalopods properly, and by inviting me aboard you made everything else possible. To the Leander Lab, especially Sarah Sparmann, thank you for your inexhaustible patience in every aspect of this thesis and life support. Thank you also to Robin Lee, and Virginia Nobel, Nobuhiko Tokuriki for your assistance with various aspects of the project. My unending gratitude goes to everyone who helped shape and support my time at UBC. To Gyan Harwood, thank you not only for your help on this project, but thank you for always supporting me through every crisis. Whether academic or not, your presence and encouragement has been invaluable.                        	 ? viii	 ?  Dedication For C.M.E.H., my rock, inspiration, and driving force.        	 ? 1	 ?Chapter 1 1.1 Introduction 1.1.1. Parasitism of cephalopods  The bizarre and charismatic animals that compose the molluscan class Cephalopoda are host to equally captivating creatures. Eukaryotic parasites can be found on and inside cephalopods in every ocean. The most heavily debated parasite lives exclusively within the renal appendage of cephalopod hosts adapted to benthic living; an enigmatic group of parasites called ?dicyemids?. These parasites ignite curiosity and bewilderment from all who encounter them, but surprisingly little is known about dicyemid biology. So much remains to be discovered and to comprehend, which makes dicyemids that much more inviting and exciting to study.   Cephalopods are found in all oceans of the world and can host a wide variety of parasites, many of which are yet to be described. Nematodes, cestodes and crustaceans infect the digestive tract of cephalopods, while apicomplexans and other microbial eukayotes are found in the gills, digestive tract and mantle (Hochberg, 1990, and Hochberg, 1982). Virulent parasites have a direct negative effect on the health of their respective host population, and with cephalopods becoming an increasingly important food source and the subject of neurological and medicinal sciences, cephalopod parasitology is likely to continue gaining importance. However, much in the area of cephalopod parasitology has yet to be pursued. Only a few species of each group have been described in current literature, leaving an opportunity for immense discovery. Some parasite species are free living during some or all of their lifecycle, while obligate parasites cannot live without a host. Some obligate parasite species have also 	 ? 2	 ?undergone the process of co-speciation, adapting to the host and mediating host adaptation (Brooks, 1979). Co-evolved parasites are restricted to a specific host species, so when the host evolves whether independently or because of the parasite, the parasite also changes to optimize adaptation (Huelsenbeck et al., 1997).  1.1.2. Dicyemid-host interactions Dicyemids (Phylum Dicyemida) are obligate parasites that live within the kidneys of benthic cephalopods (Nouvel, 1947). First discovered in 1839 by Filippo Calvolini, they were dubbed ?mesozoan? by E. Van Beneden in 1879 because they hold both metazoan and protozoan-like qualities. At the time, it was thought that they might form a bridge between microbial eukaryotes and animals. Scientists still debate if the 20-40 celled dicyemids are highly streamlined invertebrates, complex protists, or even a chimera of the two (Kobayashi et al., 1999, Noto and Endoh, 2004, Suzuki et al., 2010). Even with deeper insights afforded by molecular data, their taxonomic position still remains uncertain (Kobayashi et al., 1999, Ohama et al., 1984, Pawlowski et al., 1996, Katayama et al., 1995, Suzuki et al., 2010). Because the cephalopod diet is composed primarily of animal protein, nitrogenous waste removal becomes an important part of survival. Cephalopod metanephridia are arranged into a renal appendage composed of kidney sacs, pericardial tubules, and folds between them (Wells, 1987). Dicyemids are immersed in the host?s excreted waste and uptake the waste as nutrients via phagocytosis (Furuya and Tsuneki, 2003). Dicyemids are potentially a beautiful case of host/parasite co-speciation. Using their unusual, hard attachment apparatus called a ?calotte?, dicyemids implant in the folds and spaces of the host?s renal sacs (Figure 1.1). Dicyemid species are so specialized that 	 ? 3	 ?they are usually only found in the renal folds of one species of benthic cephalopod (Hochberg, 1990, Furuya and Tsuneki, 2003). One host can apparently house as many as four putative morphospecies of dicyemids at a time (Furuya, 1999, Furuya et al., 2003). Rarely, dicyemids have been found in pelagic cephalopods, but the occurrence is too infrequent to suggest a normal infestation (Furuya and Tsuneki. 2003).  In British Columbian waters, there are five cephalopod species that inhabit the bottom of the subtidal zone. One squid, Loligo opalescensi, and one sepiolid, Rossia pacifica are less commonly found than their relatives, the octopods. The Giant Pacific Octopus (Enteroctopus dofleini) and the Pacific Red octopus (Octopus rubescens) are the most commonly encountered.  The Smoothskin octopus (Benthoctopus levis) is also found throughout the area, though infrequently (Lamb and Hanby, 2005). 1.1.3. The life cycle and morphology of dicyemids  If dicyemids are highly specialized animals, then the adults have the fewest number of cells in almost all of the animal kingdom: the only exceptions being other parasitic species, e.g. parasitic cnidarians known as myxozoans.  There are no germ layers associated with any life stage of dicyemids (Furuya et al., 1996). Their body plan is exceptionally simplified. Vermiform individuals, the nematogens, rhombogens and vermiform embryos, all contain the same body shape and cell number. The calotte is made up of 4-10 polar cap cells that have shorter cilia than the other exterior cells on the body (Figure 1.1 and Figure 1.2). Calotte shapes are seemingly species specific and intensify niche separation (Furuya et al., 2003). Parapolar cells are positioned immediately posterior to the calotte. Uropolar cells are the most posterior cells. Trunk cells make up the rest of the 10-15 somatic cells. All the previously described peripheral 	 ? 4	 ?cells protect the axial cell, which spans the length of the body. It is thought to function solely in embryogenesis and propagation (Awata et al., 2006 and Fuyura and Tsuneki, 2003).  Dicyemids received their name from their complicated two-part life cycle. They exhibit both sexual and asexual reproduction and produce two types of larvae: vermiform and infusiform. The asexual adult phase, the nematogen, produces vermiform embryos from agametes (also known as the ?axoblast?), which are germ line stem cells embedded in the axial cell (Awata et al., 2006). Eventually, either a high dicyemid density, the sexual maturation of the host, or some unknown signal initiates the transformation of the nematogen into the sexual phase, called the ?rhombogen? (Furuya and Tsuneki, 2003).  Rhombogens are equal in cell number and body shape/size to nematogens, but infusorigens (i.e. a spherical cluster of oocytes surrounding a core of tailless spermatozoa) develop from the agamete within the axial cell instead of vermiform clones (see Figure 1.2) (Furuya et al., 2003).  Once the eggs become fertilized, they develop into infusoriform embryos through a very truncated series of spiral cleavage cell division. Nematogens produce vermiform embryos through asexual reproduction. These larval forms develop from the agamete of the adult. The cleavage starts as almost spiral, but there are only eight unequal cell division stages during embryogenesis (Furuya et al., 1996). For growth of the individual vermiform larva, cells grow in size rather than through further cell divisions (Awata, 2006).  Infusoriform larvae are reportedly the only stage to leave the host. They are faster and more agile than the vermiforms and can potentially tolerate seawater for longer. What happens to the larvae outside of the original host, potential additional life stages or 	 ? 5	 ?intermediate hosts, and how a new cephalopod host is infected remain unknown. (Figure 1.2) (Furuya and Tsuneki, 2003).  Infusoriform larvae have more cells than their vermiform counterparts, usually 37 or 39 (Furuya and Tsuneki, 2003). They are circular or oval in form with cilia on their dorsal and caudal outer cells (Figure 1.3) (Furuya, 1996). Four large urn cells surround germinal cells thought to give rise to the next generation. Infusoriforms are more complicatedly organized than vermiforms; however, they still are not considered to achieve a tissue level of organization (Furuya and Tsuneki, 2003). 1.1.4. The uncertain molecular phylogenetic position of dicyemids  Many separate lineages of parasites have evolved reduced morphological structures and genomes. The habitat of dicyemids and most endoparasites is constant and hospitable. Within the hosts? renal folds, there is no need for self-protection, foraging, digestion or complex bodily processes. Dicyemids are free from predators and have no need for costly protective behavior or body structures. They live in an environment that is constant and continually provides simple nutrients. Cephalopod waste and sperm can be easily digested at the cellular level, so having a complex digestive system could be more costly than beneficial. Osmoregulation and other regulatory processes and organs are unnecessary in such a static environment.  However, because dicyemids are so reduced in morphology, their phylogenetic position is still uncertain.  Although recent molecular phylogenetic work has done little to lessen that uncertainty, most agree that dicyemids are highly reduced animals. Where in the tree of animals they fit, however, is yet to be determined. 	 ? 6	 ? Early trees made from 5S rDNA suggested an early divergence of dicyemids from the Eumetazoa (all non-poriferan metazoans) (Omaha et al., 1984, Hori and Osawa, 1987). More recently, Kobayashi et al. (1999) discovered within a dicyemid genome both a DoxC member of the Hox-like genes and a ?spiralian? or ?Lox5? peptide; both are synapomorphies of the Lophotrochozoa.  Molecular phylogenetic analyses of 18S rDNA sequences place dicyemids within the monophyletic triploblastic animals, suggesting that embryonic germ layers have been lost. This latter gene also suggested close links with myxozoans, nematodes, and acoel flatworms (Phylum Platyhelminthes) (Figure 1.4) (Katayama et al., 1995).    The Platyhelminthes is a paraphyletic ?phylum? consisting of the Acoelomorpha, which is only distantly related to the Catenulida and the Rhabditomorpha (Halanych, 2004). The Rhabditomorpha contains two synapomorphies within their mitochondrial genomes, but the mitochondrial genome of dicyemids contains only canonical invertebrate genes and does not have either of the rhabditimorphan synapomorphies (Telford et al., 2000). This implies a divergence from either the Acoelomorpha, Catenulida, or some other point along the backbone of this paraphyletic ?phylum?.  Molecular phylogenetic analyses based on innexin amino acid sequences place dicyemids outside of the Platyhelminthes and closer to annelids or molluscs (Suzuki et al., 2010). However, because the Platyhelminthes is a paraphyletic and poorly circumscribed group, the significance of this result is hard to interpret. All of these data do have a common conclusion: dicyemids are probably triploblastic animals that have lost their embryonic germ layers and associated adult tissues. Beyond that, their phylogenetic position remains unresolved.  	 ? 7	 ?1.1.5. Dicyemid systematics  A little over a hundred (morpho)species of dicyemids have been described in 40 species of benthic cephalopods. Characterization of dicyemid genera is based on numbers of parapolar and metapolar cells found in the calotte (Figure 1.5) (Furuya, 2006). Differentiation at the species level has mainly been based on body size, cell number, calotte morphology, and host species (Furuya and Tsuneki., 2003, Furuya et al., 2003). Vermiform stages of each species have a set number of each type of outer, somatic cell. For example, Dicyema nouveli found in Loligo duvauceli is described as having 26-28 peripheral cells (i.e. 4 propolar, 4 metapolar, 2-3 parapolar, 12-14 diapolar and 2 uropolar cells) (Kalavati et al., 1984).  There are four recognized regular shapes of calottes in dicyemids: conical, cap, disk, and irregular. Up to four putative dicyemid species can be found in one cephalopod species and when multiple parasite species occur, each has a different shaped calotte than that of their co-habitants. Never have two species of dicyemid with the same calotte shape been established in a single host species (Furuya et al., 2003).   Within the commonly found Northeast Pacific cephalopods, there are eight currently described species of dicyemids. Enteroctopus dofleini hosts Dicyemennea abreida and Dicyemodeca deca. Dicyemennea abreida has 24-35 total cells, a conical-shaped calotte, and is about a millimetre in length. Dicyemodeca deca has 24-25 total cells, a disc-shaped calotte, and is also about a millimetre long (McConnaughey, 1957). The biggest differences between the two species are the calotte shape and the extra metapolar cell that Dicyemodeca species have over Dicyemennea species.  Rossia pacifica contains Dicyemennea brevicephaloides and Dicyemennea rossiae in their renal appendages. Dicyemennea brevicephaloides has 24 cells, a disc-	 ? 8	 ?shaped calotte, and can grow up to four millimetres. Dicyemennea rossiae has 30-35 cells, a conical calotte and is about two millimetres in length (Furuya, 2007). Both species have the same number of polar cells, and their differences lie mainly in body size and calotte shape.  Octopus rubescens is host to four morphospecies of dicyemids: Dicyema apollyoni, Dicyemennea adscita, Dicyema brevicephala, and Dicyema adminicula.  The calotte shapes are conical, cap-shaped, disc shaped and irregular, respectively (Furuya et al., 2003). However, usually only one, two or a maximum of three of these species are found in any one individual of this octopus species. Current assumed phylogeny based on the genus categorization of all eight morphospecies is shown in Figure 1.6.  The boundaries between dicyemid species are based strongly on calotte shape. No molecular phylogenetic data have been used to determine new species or to validate existing species, and the very little microscopy that has been performed so far has centered on light microscopy with a few instances of transmission electron microscopy (TEM) (Czaker, 2000, Ridley, 1968, 1969). No scanning electron microscopy has been reported on a whole mount dicyemid thus far.   1.1.6. DNA barcoding  DNA sequences offer a way to provide a ?barcode? to delimit species from one another. Unique strings of DNA sequences can be used as species descriptors much like barcodes are used to distinguish produce at a supermarket. The idea being that each barcoding gene has low intraspecific variation and substantially more interspecific variation (the so-called ?barcoding gap?). DNA barcodes are particularly useful for 	 ? 9	 ?determining species boundaries in organisms that have either limited morphological variation or too much intraspecific variation at the morphological level (Evans et al., 2007, Herbert et al., 2003, Moritz, 2004, Radulovici et al., 2010). Ideally, one gene would be used as the sole barcoding gene for all organisms. However, history has shown that certain genes work better for some organisms than for others. The most prevalent gene used to barcode eukaryotes is the cytochrome c oxidase subunit 1 (CO1) in the mitochondria (Evans et al., 2007, Herbert et al., 2003, Moritz, 2004). CO1 is not always suitable for all eukaryotes, so other mitochondrial markers, such as mitochondrial small subunit (16S) ribosomal DNA sequences, are also commonly used (Bucklin et al., 2010). Mitochondrial genes, which are under less pressure to remain unchanged than nuclear genes, provide the relatively fast evolutionary rate necessary to distinguish and code organisms to the species level (Palumbi and Cipriano, 1998). Yet, in parasites, mitochondria are frequently reduced or absent and amplifying these genes can be difficult (Awata et al., 2006 and Tsaousis et al., 2008). The small subunit in the nuclear ribosomal gene (SSU rDNA or 18S rDNA) has mainly been used as a phylogenetic marker because it is present in all eukaryotes, is conserved enough for constructing confident multiple-sequence alignments, and is variable enough for the elucidation of phylogenetic signal. Moreover, this marker has been known to evolve rapidly in parasites, which facilitates DNA barcoding at the species level but limits its utility for phylogenetic inference at deeper levels (i.e., more distant relationships) (Bucklin et al. 2010, Crainey et al., 2009, Floyd et al., 2002, Holterman et al., 2006, 2009, Powers, 2004). A few sequences of the 18S rRNA gene have been amplified from a few species of dicyemids (Katayama et al. 1995, Pawlowski et al., 1996, 	 ? 10	 ?Aruga et al., 2007). Therefore, amplifying additional sequences of this marker from more species is an appropriate starting point for deciphering inter- and intraspecific genetic variation in dicyemids. 1.1.7. Using molecular data to validate dicyemid species So far, species of dicyemids have been established based only on comparative morphology of traits observed with light microscopy (Furuya, 2006). Molecular phylogenetic data have helped determine the position of the Dicyemida within the tree of eukaryotes, but these data have never been used to validate generic and species boundaries within the Dicyemida (Kobayashi et al., 1999, Ohama et al., 1984, Pawlowski et al., 1996, Katayama et al., 1995, Suzuki et al., 2010). Are current morphospecies consistent with molecular phylogenetic data? My aim is to apply DNA barcoding methods to test species boundaries in dicyemids and infer phylogenetic relationships. Currently, a single cephalopod host can contain multiple cosmopolitan genera based on comparative morphology, which would imply that dicyemids are not host specific, contradicting what has been repeatedly shown. It is possible, however, that dicyemids are host specific but the morphological bases for current generic and species identification are misleading. If so, then one cephalopod species should be host to genetically similar species, and more closely related cephalopods should host more closely related parasites. Molecular phylogenetic data are expected to shed considerable light on whether or not current genera and species reflect phylogenetic relationships and whether they have coevolved with their hosts   	 ? 11	 ?Chapter 1 Figures Figure 1.1 ? Vermiform dicyemid            Adult rhombogen found in local Pacific Red Octopus Genus Species. 1) Disc shaped calotte. 2) Axial cell. 3) Truck cells. 4) Uropolar cells. Phase micrograph. Scale bare = ?m                 1	 ?2	 ?3	 ?4	 ?	 ? 12	 ?Figure 1.2 ? Known life stages in the dicyemid lifecycle    Abbreviations: AG, agamete; AN, axial cell nucleus; AX, axial cell; C, calotte; DI, developing infusoriform embryo; DP, diapolar cell; DV, developing vermiform embryo; IN, infusorigen; MP, metapolar cell; PA, parapolar cell; PP, propolar cell; UP, uropolar cell (Fig. 1, Fuyura and Tsuneki, 2003).   H. Furuya and K. Tsuneki520 atogen or rhombogen (Fig. 2a, b), and (2) the infusoriformembryo which develops from a fertilized egg producedaround the hermaphroditic gonad called the infusorigen (Fig.2c, d). The infusorigen itself is formed from an agamete. Thename ?dicyemids? is derived from the fact that they producetwo types of embryo in the life cycle. A high population den-sity in the cephalopod kidney may cause the shift from anasexual mode to a sexual mode of reproduction (Lapan andMorowitz, 1975). Vermiform stages are restricted to therenal sac of cephalopods, whereas the infusoriform embryosescape from the host into the sea to search for a new host.Infusoriform larvae actively swim  in vitro  for a few days(McConnaughey, 1951). However, it remains to be under-stood how infusoriform larvae develop into vermiform stagesin the new host. DICYEMID FAUNA IN JAPAN Dicyemids are distributed in a variety of geographicallocalities: Okhotsk Sea, Japan sea, Western and EasternNorth Pacific Ocean, New Zealand, North Indian Ocean,Mediterranean, Western North and Eastern Atlantic Ocean,Gulf of Mexico, and Antarctic Ocean (Hochberg, 1990).About 104 species of dicyemids have so far been reportedin at least 40 species of benthic cephalopods of the world.The first record of dicyemids in Japan was made by Nouveland Nakao (1938). They described  Dicyema misakiense Nouvel and Nakao, 1938 from  Octopus vulgaris  Lamarck,1798, and  D. orientale  Nouvel and Nakao, 1938 from  Sepi-oteuthis lessoniana  Lesson, 1830. Later, Nouvel (1947)described  D. acuticephalum  Nouvel, 1947 from  O. vulgaris and identified a dicyemid species from  Sepia esculenta Hoyle, 1885 as  Pseudicyema truncatum  Whitman, 1883,which had been described earlier in Europe. We have beensurveyed Japanese cephalopod and 42 species ofdicyemids including described ones have been recognizedfrom 19 species of cephalopods (Table 1). Among them, twodicyemids were described from  O. vulgaris  and  O. minor (Sasaki, 1920) as  Dicyema japonicum  Furuya and Tsuneki,1992 and  Dicyema clavatum  Furuya and Koshida, 1992,respectively (Furuya  et al ., 1992a). Later, 14 dicyemid spe-cies were described from six species of cephalopods(Furuya, 1999). A dicyemid species from  Sepia esculenta, once identified as  P. truncatum  by Nouvel (1947), differsfrom  P. truncatum  in Europe in distribution, host species,length of vermiform stages and infusoriform embryos, andthus described as a new species,  P. nakaoi .In general, dicyemids are found in benthic cephalopods,namely, octopuses and cuttlefishes. However, a few speciesof dicyemids were reported in squids,  Sepioteuthis lessoni-ana  (Nouvel, 1947) and  Loligo  sp. (Kalavati and Narasimha-murti, 1980). Such cases have been considered to be ratherexceptional. Recently, we also have found two undescribeddicyemid species from two species of squids,  S. lessoniana and  Todarodes pacificus  (Table 1). Host species ofdicyemids might not be necessarily restricted to the benthic Fig. 1. Life cycle of the dicyemids. The dashed line indicates anunknown process involved in the infection of a new cephalopod anddevelopment into adult forms. In vermiforms (nematogen, rhombo-g n, vermiform embryo), a large cylinderical axial cell is surroundedby peripheral cells. Four to ten anterior peripheral cells (propolarsand metapolars) form a calotte. The other peripheral cells are diapo-lars. Two posterior diapolars are somewhat specialized as uropo-lars. The development of infusorigens, gametegenesis around theinfusorigen and development of two types of embryo all proceedwithin the axial cell cytoplasm. AG, agamete; AN, axial cell nucleus;AX, axial cell; C, calotte; DI, developing infusoriform embryo; DP,diapolar cell; DV, developing vermiform embryo; IN, infusorigen;MP, metapolar cell; PA, parapolar cell; PP, propolar cell; UP, uropolarcell.	 ? 13	 ?Figure 1.3 ? Dicyemid infusiform larva  Found in local Pacific Red Octopus, O. rubescens. Figure 1.4 ? Relationship between the 18S sequences of dicyemids and other eukaryotes           A phylogenetic tree based on18S sequences show dicyemids diverging within triploblastic animals. (Fig. 2, Katayama et al., 1995).  88 T. KATAYAMA ET AL. Sagittu crassa Artemia salina Asterias amurensis Crassostrea gigas Shistosoma mansoni Dugesia japonica Planocera multitentaculata Moliniformis moliniformis Caenorhabditis elegans 1 I _ 40 48 100 Convoluta naikaiensis Henneguya sp. 100 Dicyema acuticephalum Anemonia sulcata Trichoplax adhaerens Beroe cucumis Diploblasts Arabidopsis thaliana Volvox carteri Hartmanella vermiformis I Plants Protists Theileria annulata Crypthecodinium cohnii I Fungi 40 Triploblasts Figure 3. The consensus tree obtained using the maximum-parsimony algorithm with bootstrap resam- pling (DNAPARS, SEQBOOT, and CONSENSE programs of PHYLIP package, version 3Sc), showing the phylogenetic position of the dicyemids among 28 repre entative eukaryotic taxa. The percentage of 500 parsimony replicates is shown at the node the value is supporting. The tree was rooted by using Saccharomyces cerevisiae as an outgroup. Branch lengths are proportional to the scale given in number of substitutions (a total of 2807). This tree is different from the three most parsimonious trees in the positions of Crassostrea gigas and Molinzformis mohniformis within the assemblage of the coelomate triploblasts. authors have proposed that the dicyemids are a missing link between unicellular organisms and multicellular an- imals (Dodson, 1956; Hyman, 1959; Lapan and Morow- itz, 1974; Ohama et al., 1984) while others have claimed that they are an animal group degenerated as a result of parasitism (Nouvel, 1948; McConnaughey, 195 1; Stun- kard, 1954; Ginetsinskaya, 1988). The phylogenetic trees inferred from comparisons of nucleotide sequences of 5s rRNA suggested that the dicyemids emerged first among the metazoa examined and that triclad flatworms, nem- atodes, cnidarians, and sponges followed, in that order (Ohama et al., 1984, Hori and Osawa, 1987). This sug- gestion does not, however, accord with the present result and the previous inferences about metazoan phylogeny based upon 18s and 28s rDNA sequences (Field et al., 1988; Christen et al., 199 1; Waim-ight et al., 1993; Ko- bayashi et al., 1993). Discrepancies are partly ascribable to differences in the methods used to infer phylogenetic relationships. In contrast to the 18s and 28s rDNA trees reconstructed by the neighbor-joining, maximum-parsi- mony, and maximum-likelihood methods, the above 5s rRNA trees have been reconstructed by unweighted and weighted pair group methods using arithmetic averages (UPGMA and WPGMA, respectively), which are valid under the assumption that rates of nucleotide substitution are constant among taxa analyzed (Sokal and Mitchener, 1958). However, the essential point is that the 5s rRNA is too small to contain signal sufficient to allow precise inference of phylogenetic relationships. Because of large standard errors, sequential orders of branching of the di- cyemids, flatworms, nematodes, cnidarians, and sponges shown in the above 5s rRNA trees appear to be statistically insignificant. Recently Halanych (199 1) analyzed the se- quence data of 5s rRNA with the maximum-parsimony method. The phylogenetic tree obtained was inconsistent with phylogenies based on 18s and 28s rDNA data, and few nodes in the tree were supported by bootstrap value at a significant level. The present results do not appear to support the prop- osition that the dicyemids are a truly primitive group linking unicellular organisms with multicellular metazoa. Instead, our results favor the view that the dicyemids are 	 ? 14	 ?  Figure 1.5 ? Pictorial description of dicyemid genera  :  Depiction of polar cells of different dicyemid genera. White blocks are parapolar cells, grey blocks are metapolar cells (from Fig.  5, Furuya, 2006).  Figure 1.6 ? Assumed phylogenetic relationship of dicyemidae genera  Basic tree of the phylogenetic relationship between local species within the Dicyemennea, Dicyemodeca and Dicyema should the current genera be correct. Japanese Society of Systematic ZoologyNII-Electronic Library Service	 ? 15	 ?Chapter 2 2.1. Synopsis  Cell number and other morphological traits have played a prominent role in identifying species of dicyemids. The validity of these traits in delimiting species, however, has never been tested with molecular phylogenetic data. If these morphological data are valid, then species representing different genera should have a greater genetic distance between them than the genetic distances between different species from the same genus. Because multiple morphospecies and genera can be found within a single cephalopod host, a comparison of 18S rDNA sequences derived from specimens representing these species and genera will allow me to evaluate if current morphology-based descriptions are accurate reflections of dicyemid species diversity. Patterns of genetic distances between different 18S rDNA sequences are also expected to help elucidate patterns of co-evolution between dicyemids and their hosts. For instance, did dicyemids codiversify with their hosts, that is closely related species tend to be associated with one host species?  Or are dicyemid species capable of infecting many different host species, so that more distantly related dicyemid species tend to be found within one host species? I chose to explore the variation of 18S rDNA sequences to test the validity of current dicyemid species and genera because this marker (1) is known to be rapidly evolving in parasitic eukaryotes in general, (2) has been amplified previously from a few other species of dicyemids, and (3) has been used successfully as a DNA barcode in other groups of parasites (e.g., nematodes and apicomplexans) (Bucklin et al., 2010, Crainey et al., 2009, Floyd et al., 2002, Holterman et al., 2006, Holterman et al., 2009, Kobayashi et al., 1999, Katayama et al., 1995, Pawlowski et al., 1996, Powers, 2004, and Tsaousis et 	 ? 16	 ?al., 2008). 18S rDNA sequences were amplified from the following currently recognized taxa: Dicyema apollyoni, Dicyema brevicephala, Dicyema adminicula, and Dicyemennea adscita from the Pacific Red Octopus (Octopus rubescens); Dicyemennea brevicephaloides and Dicyemennea rossiae from the Stubby Squid (Rossia pacifica); and Dicyemodeca deca and Dicyema abreida from the Giant Pacific Octopus (Enteroctopus dofleini). My results demonstrated that 18S rDNA sequences of individual dicyemids representing several different morphospecies collected from the same host were nearly identical. These data suggest that each host was infected by a single dicyemid species rather than several different species that reflect variations in morphology. I also attempted to test the validity of the 18S rDNA sequence data with additional molecular markers that are known to be rapidly evolving, namely CO1, mitochondrial 16S rDNA, and the nuclear? Internal Transcribed Spacers (ITS). Although I was not able to determine reliable primers for these markers within the time constraints of this MSc study, combining these data with the 18S rDNA sequences is expected to provide a powerful way to demonstrate species boundaries within dicyemid parasites.  2.2. Methods  2.2.1. Collection of specimens and light microscopy  Host cephalopods were collected in collaboration with spot prawn fishermen of BC Spot Prawns, captained by Rick Jerema (bcprawns.gmail.com) off the Sunshine Coast in British Columbia, Canada. Twenty-five octopods, Enteroctopus dofleini and Octopus rubescens and one stubby squid (Rossia pacifica) were caught in prawn traps between May 27th and June 4th 2012 in the Jervis (50.0?N, 123.9?W) and Seshelt Inlets (49.7?N, 	 ? 17	 ?123.9?W). The visceral masses were removed, placed in tubes or bags over ice and brought back to the lab. Hosts were identified to species level using both morphological traits and CO1 sequences.   Once in the lab, the ?kidneys? were extracted from each sample and placed in ?Dicyemid Isolation Buffer? (DIB) (Lapan and Morowitz, 1975). Dicyemids were isolated under light microscopy via micropipette and placed in autoclaved seawater. From each host, sixteen dicyemids representing 2-4 different morphospecies were photographed with differential interference contrast (DIC) using a Zeiss Axiovert 200 light microscope connected to a Pixelink-A662 digital camera and then deposited into a 0.2 ml PCR tube with 10 ?l of autoclaved water. Micrographs were used to identify individual dicyemids to one of the currently recognized morphospecies found in the host species.   2.2.2. Host DNA extraction, PCR, cloning, and sequencing  DNA was extracted from the host kidney using Quiagen DNeasy? Blood & Tissue Kit. CO1 sequences were amplified with the forward primer 1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and reverse primer 2198 (5'-TAAACTTGAGCCTGACGAAAAAAATC-3') (Folmer et al., 1994). PCR samples were prepared with 12.5 ?l EconoTaq? DNA Polymerase, 10.5 ?l autoclaved distilled water, 0.5 ?l forward primer, 0.5 ?l reverse primer, and 1 ?l of extracted template DNA. The PCR samples were held at an initial denaturation period (94?C for 5 min) then 40 cycles of denaturation (92?C for 1 min), annealing (40?C for 1 min), elongation (72?C for 1 min) and a final elongation period (72?C for 5 min). PCR products were then cloned using the Agilent Technologies StrataClone PCR Cloning Kit. Purified DNA was 	 ? 18	 ?sequenced in both directions from eight clones per individual host using ABI Big-Dye? reaction mix and the cloning primers.  The new DNA sequences from the host samples were identified using BLAST. 2.2.3. Dicyemid DNA extraction, PCR, cloning, and sequencing DNA was extracted from the dicyemid isolates using the Biotechnologies Epicentre MasterPure? Complete DNA & RNA Purification Kit and stored in 35 ?l of TE buffer. 18S rDNA sequences were PCR amplified using Illustra? PuReTaq? Ready-To-Go? PCR beads, 23 ?l autoclaved distilled water, 1 ?l of extracted template DNA, and 0.5 ?l of each of the following primers: F3 (5'-CGGCTCATTAAATCGGACATAC-3') and R2 (5'-CCAACAACCTCACCAAATCATTC-3'). The PCR protocol involved an initial denaturation period (94?C for 2 min), 40 cycles of denaturing (94?C for 45 sec), annealing (50?C for 45 sec), and elongation (72?C for 2 min), and a final elongation period (72?C for 5 min).  These PCR products were then diluted to 1 in 10 parts water and used as the template for two different semi-nested PCR amplifications.  The first reaction used primers F3 and R3 (5'-CACTGTGTTCGGCCCGGGTGAG-3'); the second reaction used primers F2 (5'-GTGGATTAGATCTCGTCGTAG-3') and R2. All primers were chosen from an alignment of all dicyemid 18S sequences in GenBank (Benson et al., 2005, Katayama et al. 1995, Pawlowski et al., 1996, Aruga et al., 2007). The PCR program for these reactions was the same as described above except the 40 cycles were reduced to 25 cycles. Purified PCR products were directly sequenced in both directions using ABI Big-	 ? 19	 ?Dye? reaction mix with the amplification primers.  The new DNA sequences from the dicyemid samples were identified using.  2.2.4. Sequence alignments and molecular phylogenetic analyses:  Thirty-four new DNA sequences from dicyemids were analyzed and edited using Sequencher? before being aligned with the web-based MUSCLE (i.e. multiple sequence alignment with high accuracy and throughput) (Edgar, 2004). In addition, three available 18S rDNA sequences from dicyemids were downloaded from GenBank: Dicyema acuticephalum, a parasite of Octopus vulgaris collected from Japan; Dicyema orientale, a parasite of Sepioteuthis lessoniana collected from Japan; and Dicyema sp. collected from a Sepia officinalis in the Mediterranean Sea (Benson et al., 2005, Katayama et al., 1995, Pawlowski et al., 1996). These three sequences plus the new ones generated here formed a 37-taxon alignment that was edited by eye using MacClade (Maddison and Maddison, 2005). Indels and ambiguously aligned positions were excluded from the alignment, resulting in 1,245 unambiguously aligned sites. The NEXUS file was submitted to RAxML to construct a Maximum Likelihood (ML) tree with 100 bootstrap inferences, 0.804391 invariable sites, and an alpha parameter of 1000.0 (Stamatakis, 2006). A genetic distance matrix was completed using PAUP version 4 (Swofford, 2002).  2.2.5. Scanning electron microscopy: Individual dicyemids were placed on coverslips coated with poly-L-lysine, pre-fixed with 5% (v/v) glutaraldehyde, washed with sodium cacodylate buffer, and post-fixed with 1% (w/v) osmium tetroxide. The dicyemids were then dehydrated with a graded series of ethanol concentrations. Dicyemids in 100% ethanol were transferred into a Tousimis Supercritical Autosamdri 815B Critical Point Drier and dried with CO2 before 	 ? 20	 ?being coated with an even 5-10 nm layer of gold in a Cressington High Resolution Sputter Coater. All images were taken with a Hitachi S4700 scanning electron microscope.   2.3 Results  2.3.1 Dicyemid species collected Dicyemids representing all eight morphospecies previously recorded from Octopus rubescens, Enteroctopus dofleini, and Rossia pacifica were found in my samples: Dicyema apollyoni, Dicyema adminicula, Dicyema brevicephala, Dicyemennea adscita, Dicyemennea abreida, Dicyemodeca deca, Dicyemennea brevicephaloides, and Dicyemennea rossiae. Three Dicyema apollyoni, three Dicyema adminicula, four Dicyema brevicephala, and nine Dicyemennea adscita sequences were collected from three O. rubescens individuals.  Six Dicyemodeca deca and five Dicyemennea abreida sequences came from one E. dofleini, while five Dicyemennea brevicephaloides and four Dicyemennea rossiae sequences were collected from one R. pacifica (Table 2.1, Figure 2.1 and 2.2).  2.3.2 Molecular phylogeny analyses The 14 dicyemids collected from three different individuals of O. rubescens had identical 18S rDNA sequences; these dicyemids represented the morphotypes of Dicyemennea adscita, Dicyema adminicula, Dicyema brevicephala, and Dicyema apollyoni.  The 18S rDNA sequences derived from 11 dicyemids collected from E. dofleini, which represented the morphotypes of Dicyemodeca deca and Dicyemennea abreida, were identical to each other and 0.32% different from the dicyemid sequences collected from O. rubscens. The nine dicyemids collected from R. pacifica, which 	 ? 21	 ?represented the morphotypes of Dicyemennea brevicephaloides and Dicyemennea rossiae, were identical and 4.4% different from the dicyemid sequences collected from E. dofleini and O. rubescens (Figure 2.3).  The molecular phylogenetic analyses of 18S rDNA sequences demonstrated a polyphyletic distribution of isolates representing the current genera of dicyemids. Representatives of Dicyema, for instance, were nested within three different clades (Figure 2.3). Of the three dicyemid sequences found in GenBank, Dicyema orientale, grouped near the Dicyemennea brevicephaloides/Dicyemennea rossiae clade while Dicyema acuticephalum and an undescribed Dicyema species diverged from the same lineage as the E. dofleini and O. rubescens clades.   The two clades of dicyemid species from octopods, namely (1) Dicyemennea adscita, Dicyema brevicephala, Dicyema adminicula, and Dicyema apollyoni from O. rubescens, and (2) Dicyemodeca deca and Dicyemennea abreida from E. dofleini formed a monophyletic group to the exclusion of a clade of dicyemid species from teuthoids (Figure 2.3).  2.3.3 Scanning electron microscopy  Parasites pose a particular challenge for scanning electron microscopy (SEM). Endoparasites tend to embed within the body cavities of their hosts; for example, intestinal parasites are among the gut and organ contents of their host, making it difficult to prepare a clean SEM sample of the parasite?s exterior surface. Dicyemids live within the renal folds of their cephalopod hosts, feeding on the surrounding particles by phagocytosis (Furuya et al., 2003). The host tissue, and possibly secretions associated with it, were a problem not foreseen before the stubs were completely processed. In my 	 ? 22	 ?samples, a web of unknown substance obscured the exterior surface of the dicyemids. However, the calotte shape of the different species was still visible. Because the same information is readily seen by light microscopy, the SEM images added no new information.  2.4 Discussion: 2.4.1 Phylogeny  The 18S rDNA sequences of dicyemids show higher similarity between species within a host than species within currently recognized genera. Coevolution between host and parasite is a larger adaptive pressure than previously recognized. The molecular phylogeny of collected individuals varies greatly from the historically assumed phylogeny (Figure 1.6). My results show clearly that all three genera are polyphyletic. The phylogenetic relationships of dicyemids are strongest within a host species and within host ?families?, suggesting that dicyemids codiversified with their cephalopod hosts.   The tree inferred from 18S rDNA sequences shows the expected results of parasites co-speciating with their hosts. My results suggest that the eight previously recognized morphospecies and three previously recognized genera studied here actually represent only three different phylogenetic species with high levels of intraspecific morphological variation. This phylogenetic context is therefore critical for understanding dicyemid diversity and evolution.  The 18S rDNA sequence data we present here is very straightforward and leaves little room for interpretation. However, because we have only shown a phylogeny 	 ? 23	 ?inferred from one gene, the relationships between the dicyemid isolates may not be as clear with additional sources of data. 18S rRNA has been shown to be a rapidly evolving gene, especially in parasites (Holterman et al., 2006, Holterman et al., 2009, Powers, 2004), but because there are so few samples of dicyemid 18S rDNA sequences, it is impossible to say for certain if the marker is reliable. Additional genes like 16S rRNA, CO1 or ITS may prove to more reliably show relationships between species of dicyemids. However, it is unlikely that the results will vary greatly from the 18S rDNA data. Even in the most conserved gene, different species of dicyemids found within the same species of cephalopod host are unlikely to have identical 18S rDNA sequences. It may even be more difficult to determine species boundaries with more rapidly evolving genes (e.g., CO1) because the sequences may be too variable and impossible to align (Schloss, 2010). Further complicating the issue, adult dicyemid mitochondria is highly reduced and only expressed in a few somatic cells, making it extremely hard to amplify and sequence (Awata et al., 2006).     It is possible that COI, 16S, or ITS sequences will show variation of dicyemid organisms within a cephalopod. There could be multiple highly related dicyemid species that cohabitate a single kidney, but I doubt that they would fall into the categories currently given to them. The current phylogeny of dicyemids in the literature will not be validated using either COI, 16S, or ITS, but there could still be some unknown variations that those genes display (e.g., cryptic species). It is unlikely that there is less variation than the 18S rDNA suggests, since it is unlikely that either mitochondrial genes or ITS regions in the ribosomal RNA operon will be more conserved than one of the nuclear ribosomal genes.  	 ? 24	 ? Increasing the number of dicyemid 18S rDNA sequences available in the literature will help to disentangle the phylogenetic position of dicyemids within the tree of animals and the overall diversity of dicyemid species. Including the data presented in this thesis, there are currently only 9 different 18S rDNA sequences that reflect dicyemid species diversity. There is greater than a 6% difference between two species of dicyemids; Dicyema orientale and Dicyema acuticephalum. Similar to how long-branch attraction can be solved with an increase in the number different sequences, an increase in dicyemid sequences in phylogenetic analyses increases the chance of closing gaps between dicyemids and their closest extant relatives.  2.4.2 Specificity and Coevolution  Dicyemid coevolution with their hosts is not unexpected. Parasite-host coevolution is a well-documented phenomenon found in parasite-host relationships across the tree of animals and beyond. Many hypotheses, including The Red Queen Hypothesis, have been proposed to explain this coevolutionary relationship. In fact, Farenholz' Rule states that parasite phylogeny mirrors host phylogeny (Brooks, 1979).  Parasites both drive change in their hosts and simultaneously change in conjunction with their evolving host. The association can be so intimate that fossilized whale barnacles, for instance, have been used to make inferences about the behavior of their prehistoric cetacean hosts (Dominici et al., 2011). Monogenetic flukes have also been used to distinguish different cryptic species of host cyprids (Lambert and Gharbi, 1995). A close evolutionary relationship usually signifies host specificity of the parasite. Specificity is an adaptive strategy to maximally exploit a single host species instead of generally exploiting a wide range of host species (Brooks, 1979, Kuris et al., 1979, 	 ? 25	 ?Poulin et al., 2011). There are benefits to each strategy depending on a variety of factors, but it is most likely that dicyemids are highly host specific.  There are three main categories of specificity, structural (or basic), phylogenetic, and geographic. Structural specificity refers to how many host species one parasite species inhabits and how equally distributed the parasite population is between different hosts. If one species of parasite occupies two species of host equally, and one parasite species occupies two host species, but is found more heavily in one species than the other, then the second parasite species is more structurally specific (Poulin et al., 2011). Dicyemids can be considered to be a highly structurally specific lineage of parasites since only a few known species are found in multiple host species.   Phylogenetic specificity refers to a propensity for a parasite species to infest host species that are closely related to each other. For example, if one species of parasite infests only hosts from within the family Hominidae but another infests mammals over many families, the first is more phylogenetically specific (Poulin et al., 2011). Because dicyemids usually only infest one host species at a time, it is difficult to comment on the phylogenetic specificity of dicyemid species. However, with our molecular phylogenetic tree, we have shown phylogenetic specificity within the Dicyemida as closely related dicyemid species infect closely related hosts.   Geographic specificity relates to host specificity of a parasite species over distance. If a species infests the same hosts in one location as it does in all locations where the host is found, then it is more specific than a parasite species that switches hosts over locations (Poulin et al., 2011). Dicyemid species rarely change hosts over 	 ? 26	 ?geographic distance (if ever) so they can be considered to show high geographic specificity.     The historical classification system for dicyemid diversity showed very little phylogenetic specificity. Supposedly, closely related dicyemids were infecting cephalopods from highly divergent ?families? or ?orders?. To be so host specific in structural and geographic terms, but so general in phylogenetic terms is counter-intuitive because phylogenetic specificity is the most common form of parasite-host specificity. Random host selection is far less likely than infection by a parasite of phylogenetically related host species (Krasnov et al., 2004, Poulin et al., 2011).  The 18S rDNA sequences suggest that (1) Dicyema brevicephala, D. adscita, Dicyema adminicula, and Dicyema apollyoni are all one species; (2) Dicyemodeca deca and Dicyemennea abreida are one species, and (3) Dicyemennea rossiae and Dicyemennea brevicephaloides are one species. The morphological differences between the previously recognized species would then be interpreted as intraspecific variation.   Host specificity shows a highly complicated and intimate relationship between the two organisms. Parasites have a steady, predictable habitat in their specific hosts. As such, they can synchronize with their host so tightly that any small change made in the host selects for changes in the parasite and vice versa so any large evolution occurring over time has been mirrored in the partner symbiont.  Generalist do not have such an advantage since they have to be infectious to a wide range of habitats and cannot afford to change every time one of their hosts does.  This synchronicity of host and parasite leads to what we see as cospeciation between the two. Because dicyemids are so specialized both structurally and 	 ? 27	 ?geographically, the data we present is not surprising. To fully exploit one species of host, a parasite species must occupy all available niches within its environment. Throughout the generations of cephalopod renal inhabitation, dicyemid species encountered different types of space within the renal fold. These different spaces could be best exploited with one of four basic shapes of calottes. My 18S rDNA sequence data suggest that these basic calotte shapes are convergent features, evolving multiple times as each dicyemid species adapted to variations in host physiology. It is also possible dicyemid individuals are capable of adopting any suitable calotte shape during development. It is not unusual for individuals of a species to change morphology to fill separate niches (Nolte et al., 2010).   For the hundreds of years that dicyemids have been known to scientists, emphasis has been placed on describing new species. Over a hundred dicyemid species have been described in under 50 species of cephalopod (Castellanos-Martinez et al., 2011). My results suggest that the actual number of described dicyemid species is much smaller. Furthermore, of the over 600 species of benthic cephalopods, very few probably harbor more than one species of dicyemid. By using molecular tags as species identifiers, researchers will quickly be able to identify new dicyemid species in unexplored hosts as well as collapse multiple morphological species into fewer phylogenetic species. I would predict that ultimately there are 600-700 species of dicyemids worldwide. 2.4.3 SEM images   I was not able to sufficiently correct preparation problems with unknown material covering the cilia of the dicyemids. All preparation of all individuals were completed before I could look at the results under the SEM, so any failure in the preparation were not found and corrected before all the specimen were permanently fixed. In the future, 	 ? 28	 ?individuals should be washed several times repeatedly before fixation and if possible, collection and sample preparation should be spread out over time to avoid the same predicament. Chapter 2 Figures Figure 2.1 ? Pictorial descriptions of local dicyemid morphospecies  Dicyemid species found in local cephalopods. 1-4 From O. rubescens. 1 Dicyemennea adscita (Fig. 1(m) in Furuya et al., 2003) 2 Dicyema apollyoni (Fig. 1(l) in Furuya et al., 2003) 3 Dicyema adminicula (Fig. 1(o) in Furuya et al., 2003) 4 Dicyema brevicephala (Fig. 1(n) in Furuya et al., 2003). 5, 6 From E. dofleini. 5 Dicyemodeca deca (Fig. 1(a) in McConnaughey, 1957) 6 Dicyemennea abreida (Fig. 3(d) in McConnaughey, 1957). 7, 8 From R. pacifica. 7 Dicyemennea brevicephaloides (Fig. 3(c) in Furuya 2007) 8 Dicyemennea rossiae (Fig. 5(e) in Furuya 2007).              	 ? 29	 ?        Figure 2.2 ? Local dicyemid isolates  Dicyemid individuals isolated from cephalopods collected near Jervis Inlet and Sechelt, BC. 1-4 From O. rubescens. 1 Dicyemennea adscita as sequenced. Scale bar = 10 ?m. 2 Dicyema apollyoni as sequenced. Scale bar = 30 ?m. 3 Dicyema adminicula as sequenced. Scale bar = 10 ?m. 4 Dicyema brevicephala as sequenced. Scale bar = 7 ?m. 5, 6 From E. dofleini. 5 Dicyemodeca deca as sequenced. Scale bar = 10 ?m. 6 Dicyemennea abreida as sequenced. Scale bar = 10 ?m. 7, 8 From R. pacifica. 7 Dicyemennea brevicephaloides as sequenced. Scale bar = 30 ?m.  8 Dicyemennea rossiae as sequenced. Scale bar = 40 ?m.    	 ? 30	 ?Figure 2.3 ? ML tree of all current dicyemid 18S sequences  Maximum Likelihood phylogenetic tree of all dicyemid 18S rDNA sequences used in this study: 34 new dicyemid sequences and 3 dicyemid sequences from GenBank representing 8 morphospecies. The host species for each dicyemid species is labeled in black to the right of the dicyemid individuals. Dicyemids from octopods are shaded in pink; dicyemids from teuthoids are shaded in yellow. Dicyemids from the morphology-based genera Dicyema, Dicyemennea, and Dicyemodeca are shown in red, blue, and green font, respectively. Bootstrap values of each node are shown in black. Branch lengths represent the mean number of nucleotide substitutions per site.     	 ? 31	 ?Chapter 2 Tables Table 2.1 ? Dicyemids and hosts collected  Host Species Collected Dicyemid Species Sequenced     O. rubescens 3 Dicyema apollyoni 3 Dicyema brevicephala 4 Dicyema adminicula 3 Dicyemennea adscita 9     E. dofleini 1 Dicyemodeca deca 6 Dicyemennea abreida 5     R. pacifica 1 Dicyemennea brevicephaloides 5 Dicyemennea rossiae 4   Dicyemid species collected and sequenced and the host cephalopods in which they were found.         	 ? 32	 ?Chapter 3  3.1 Conclusion: 3.1.1 Status of species The main aim of this study was to determine whether or not morphological and molecular traits accurately reflect the boundaries between different species of dicyemids in order to better understand how these parasites evolved with their cephalopod hosts. Determining species boundaries, however, is rarely straightforward. There are multiple species concepts that different factions of scientists have argued over for decades (Mayden, 1997). The most widely accepted concept for animals is the Biological Species Concept (BSC), which defines a species as a group of individuals capable of mating with one another to produce fertile offspring. This species criterion cannot be pragmatically applied to dicyemids because they are only known to self-fertilize. We must then use other methods to determine species boundaries in this group of parasites. Previous dicyemid biologists have relied on the Morphological Species Concept (MSC) (closely related to the Typological Species Concept) to describe the diversity of the group; the MSC considers species as ?the smallest groups that are consistently and persistently distinct, and distinguishable by ordinary means? (Cronquist, 1978, Mayr, 1976). This concept makes sense in pragmatic terms and allows a person of limited training to separate species from one another; however, this approach has been shown to be misleading in many instances when tested with molecular data and is incapable of revealing cryptic species, hybrids, and variation (or the lack their of) caused by different rates of morphological trait evolution. The MSC is commonly regarded as problematic and insufficient for use on its own to describe species boundaries (Donoghue, 1985, Mayden, 1997, Mayr, 1975, and Simpson, 1951). 	 ? 33	 ?The remaining concepts that are most applicable to the dicyemid species debate are the Ecological Species Concept (EcSC), Evolutionary Species Concept (ESC) and Phylogenetic Species Concept (PSC) (Mayden, 1997). The EcSC and ESC are almost synonymous: the EcSC considers species to be ?a lineage (or closely related set of lineages) which occupies an adaptive zone minimally different from that of any other lineage in its range and which evolves separately from all lineages outside its range?; and the ESC considers a species to be ?a lineage (an ancestral-descendant sequence of populations) evolving separately from others and with its own unitary evolutionary role and tendencies? (Mayden, 1997, Van Valen, 1976, Simpson, 1951). If we take only these definitions into account, then all dicyemid morphospecies living concurrently within a host species would be considered one single species. The molecular data I gathered in this study are consistent with these concepts because all of the sampled morphospecies isolated from a single host species had identical 18S rDNA sequences. My results are also consistent with the PSC, which considers separate species to be the monophyletic groups of lineages near the tips of a phylogenetic tree (Donoghue, 1985).  Altogether, the molecular phylogenetic data demonstrated that current dicyemid species based on the MSC are invalid, and the actual species diversity of dicyemids is consistent with the EsSC, ESC, and PSC. 3.1.2 The strengths and weaknesses of molecular phylogenetic data  Technological advances have increased the speed and efficiency of molecular phylogenetic methods but there are important pitfalls associated with the analysis of DNA sequence data. Long-branch attraction (LBA) artifacts are also important problems with DNA sequence data when two or more sequences are highly divergent from the 	 ? 34	 ?majority of the sequences in an alignment. Each branch length signifies evolutionary changes with the longest branch signifying the greatest change from the ancestral state. The fast-evolving sequences cluster together not out of similarity to each other, but because of shared dissimilarity to the rest (Bergsten, 2005). Although this phenomenon is a problem when comparing dicyemids to other organisms, LBA is not problematic when comparing different dicyemid species. Ultimately, phylogenies are working hypotheses that can be tested and modified as improved methods or additional data become available (Nadler, 1995). One of the primary advantages of molecular data is the relatively large number of heritable characteristics that can be compared in phylogenetic analyses (Hillis, 1987). The data can also be archived and accessed very efficiently. GenBank, for instance, has made millions of annotated DNA sequences readily available to the global scientific community (Benson et al., 2005). DNA sequences also offer the most promising data to objectively and accurately delimit different species of organisms from one another, especially parasitic species like dicyemids. DNA sequences are particularly helpful in (1) identifying morphologically similar but genetically divergent (cryptic) species and (2) morphologically divergent but genetically similar species. The interpretation of molecular data can also be much more straightforward than the interpretation of morphological data. Organisms that previously took years of experience to distinguish by a few experts can now be readily identified in a matter of days by a much larger pool of people (Powers, 2004).  	 ? 35	 ?            3.1.3. Dicyemid diversity in the age of molecular biology To this day, the life history of dicyemids is perplexing. There are multiple stages to the lifecycle that may not be grouped together by morphology alone. Of known dicyemid morphospecies, four life stages are generally described, but to varying completeness. Descriptions of infusoriform larvae can be confusing or non-existent. Having DNA barcodes in place can link all stages of a species? life cycle, filling in the gaps left by morphological methods. DNA barcoding is applicable to all life stages; a barcode of an individual remains the same throughout its entire lifecycle, so a nematogen?s barcode sequence will be identical to the infusoriform larva?s barcode sequence (Powers, 2004).  At present, there is an obvious gap in the known life cycle of dicyemids. There are no clues about what happens to an infusoriform larva once it leaves the host.  The larva could directly infect another cephalopod, or there could be more life stages yet to be discovered. It is possible that another stage has been found already but due to inconsistent morphology, has been classified within a separate lineage of organisms altogether (e.g., another ?phylum?). Through DNA barcoding, dicyemids can be easily linked to their potential missing counterparts, closing one of the largest gaps in dicyemid knowledge.  By bringing 21st century technology to dicyemid biology, I have been able to use genetic information to test species boundaries and determine a more accurate description of host-parasite relationships. Because this is the first study that set out to determine species boundaries with molecular markers, more genes should be examined to solidify these conclusions. However, I anticipate the new data will confirm that the 1,200 bp of the 18S rRNA gene examined in this study provide enough variation to separate true species while having the added advantage of being useful in determining deeper 	 ? 36	 ?phylogenetic relationships (Powers, 2004, Powers et al., 2011). The availability of primers and the relative ease of amplification made 18S rDNA sequences of dicyemids a natural starting point for DNA barcoding. 18S rDNA sequences have also been used as a barcode gene several times by previous parasitologists because this gene tends to be highly divergent in parasites (Nadler, 1995, Powers, 2005, Powers et al., 2011). However, this molecular marker is not the most commonly advocated gene used in DNA barcoding of other organisms, especially animals. The strength of the 18S rRNA gene as a DNA barcode in dicyemids (and other parasites) can be tested by juxtaposing these data with even more rapidly evolving sequences, such as the Internal Transcribed Spacer (ITS), the mitochondrial cytochrome oxidase I (COI), and the mitochondrial 16S rRNA gene. Both COI and ITS have been shown to be effective barcoding genes in certain groups of eukaryotes. COI is the most frequently used barcoding gene among animals and ITS and COI are also widely used among microbial eukaryotes  (Bucklin et al., 2011, Herbert et al., 2003, Mitani, et al., 2009, Ratnasingham and Hebert, 2007, Schoch et al., 2012). Higher variation in the sequences increases the information we can infer of evolutionary history among closely related organisms; however, too much variation obliterates phylogenetic signal between more distantly related organisms due to an increase in random signal. The mitochondrial gene COI and ITS, an intron, have a low evolutionary pressure for sequences to remain stable. This makes them highly useful genes as DNA barcodes. The conclusions in this thesis based on 18S rDNA sequences can be confirmed or modified by generating sequences of COI and ITS from the same dicyemid morphotypes. If the ITS or COI sequences show a higher amount of variation among the dicyemid morphospecies than the 18S rDNA sequences, then new meaningful 	 ? 37	 ?boundaries between lineages might be revealed. Nonetheless, the enigmatic dicyemids offer an interested phylogenetic puzzle, and the more pieces we unearth, the clearer the picture about their overall diversity and evolutionary history we can achieve. Technical difficulties and future directions Many unsuccessful attempts to uncover COI sequences from both the isolated dicyemids and the intact cephalopod containing dicyemids were made. Three COI pairs of primers were used on the dicyemid DNA. Two pairs were taken from previous studies that were able to successfully amplify and sequence dicyemid COI (Awata et al., 2006, Watanabe et al., 1999). Only the second pair produced the one sequence that is available on GenBank (Watanabe et al., 1999). The third pair used was a general universal pair of COI primers that have been used to successfully amplify sequences of both protists and animals (Folmer et al., 1994).  No 16S rDNA sequences from dicyemids had been made available, so ubiquitous animal primers were attempted.  Each primer set was attempted several times with a range of annealing temperatures from 40-52?C. Only cephalopod sequences were ever retrieved. Attempts to amplify 16S rDNA and both ITS sequences from isolated dicyemid DNA were equally unsuccessful. Two pairs of primers were repeatedly attempted for 16S rDNA amplification; both were given annealing temperatures between 50-60?C. Both were standard universal primer pairs for all animals (Mitani, et al., 2009, Sparmman unpublished). The forward primer for ITS1 (between the 18S rDNA and 5S rDNA genes) was custom made from the previously acquired sequences. One 5S rDNA sequence was previously produced, and two attempts were made to create primers from alignments containing that sequence (Ohama et al., 1984).  There is no sequenced 28S rDNA for 	 ? 38	 ?dicyemids so primers were assembled using alignments of representatives from every major animal lineage. Universal platyhelminthes ITS primers were also attempted (Kr?lov?-Hromadov? et al., 2008). The closest sequences retrieved were of fungi and bacteria. After several months of failed attempts, it was concluded that further effort was unlikely to produce results. With unlimited time and resources, the correct combination of annealing temperature and primer pair could be uncovered, but when that will occur is unknown. Full genome sequencing is quickly becoming more affordable and efficient. At the moment, the work involved is still too involved and costly to be practical for a MSc project, but is not wholly unfeasible and could be the next step to tackle the dicyemid species problem. In addition, more dicyemid morphospecies should have their 18S rRNA genes sequenced in order to demonstrate broader phylogenetic patterns within the group. Because each cephalopod species that I studied hosted parasites with unique sequences, the data suggest that 18S rDNA sequences offer the phylogenetic signal needed to distinguish different species of dicyemids from one another. With more sequences of dicyemids from a larger sampling of host species from around the world, the 18S rDNA marker may prove to be a reliable DNA barcode. Some cephalopods may indeed host multiple species of dicyemids. However, my results suggest that many more dicyemid species will prove to be invalid to uncovering more clues to dicyemid evolutionary history.     	 ? 39	 ?Works Cited  Abbott, N. J., Williamson, R., & Maddock, L. (1995). Cephalopod neurobiology: neuroscience studies in squid, octopus, and cuttlefish. Oxford: Oxford University Press.  Aruga, J., Odaka, Y. S., Kamiya, A., & Furuya, H. (2007). 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