NUCLEAR, PLASTID AND MITOCHONDRIAL GENES FOR DNA IDENTIFICATION,

BARCODING AND PHYLOGENETICS OF APICOMPLEXAN PARASITES

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

JOSEPH DAIRO OGEDENGBE

In partial fulfillment of requirements

for the degree of

Doctor of Philosophy

May, 2011

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NUCLEAR, PLASTID AND MITOCHONDRIAL GENES FOR DNA IDENTIFICATION, BARCODING AND PHYLOGENETICS OF APICOMPLEXAN PARASITES

Joseph Dairo Ogedengbe Advisor: University of Guelph, 2011 Dr. J.R. Barta

The consists of parasitic organisms that are of major health and economic importance to man and other especially domestic ones. The systematics of these parasites has received some attention in recent years but there are still many areas of study that need elucidation. Morphological data has been shown to be insufficient in answering most phylogenetic questions especially within the Emerioriniid which is the main focus of this study. The need to corroborate and in some instances modify classical systematics with molecular data is a major thrust of phylogenetic studies. Most studies have however focused on limited number of genomes and genes and have led to new challenges in the molecular phylogenetics of the phylum Apicomplexa. In this study, nuclear as well as organellar genomes were explored both as molecular identification tools as well as markers for molecular phylogenetic studies. It is concluded that mitochondrial genes such as cytochrome oxidase c I is a useful diagnostic (barcoding) and corroborative molecular tool and that organellar (both plastid and mitochondrial) genes in conjunction with nuclear genes in a combined, robust phylogenetic analysis coupled with extensive taxon sampling offer a useful approach to understanding phylogenetic relationships within the Apicomplexa. ACKNOWLEDGEMENTS

I am indebted and grateful to have worked with my advisor, Dr John R. Barta for his insights, encouragement and constant support from the conception of this work to its completion.

I would also like to thank my graduate committee members; Drs Robert H. Hanner;

Patrick Boerlin; and Bruce Hunter for their support, interest and suggestions to this

Thesis.

I would also like to thank Dr. Katarzyna B. Miska for accepting the task of being my external examiner.

I wish to express my sincere thanks to Julie Cobean for her kind provision and untiring efforts in making available pure isolates of most of the eimeriid coccidia used for this study. The contributions of Dustin Curts to DNA isolation from field isolates of coccidia are acknowledged.

I thank the National Veterinary Research Institute, Vom Nigeria, NSERC National

Science and Engineering Research Council of Canada (NSERC), and the Ontario

Ministry of Agriculture, Food and Rural Affairs (OMAFRA) for funding this study.

I would last but not the least thank all members of my family especially my wife,

Mosunmola and children for bearing with my long absence and their encouraging words when I needed it the most.

1 TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF TABLES iv LIST OF FIGURES v LIST OF APPENDICES vi LIST OF ABBREVIATIONS vii Declaration of Work 1 Chapter I: Introduction and Literature Review 2

1.1. INTRODUCTION 2 1.2. THE PHYLUM APICOMPLEXA 4 1.3. METHODS OF IDENTIFICATION 9 1.4. MOLECULAR PHYLOGENETICS 11 1.4.1. Molecular Phylogeny of the Apicomplexa (Levine, 1970) 16 1.5. MOLECULAR MARKERS AND GENOMES USED IN APICOMPLEXAN PHYLOGENY 31 1.5.1. Nuclear small subunit gene (18S rRNA gene) 31 1.5.2. Mitochondrial genome 33 1.5.3. Plastid genome 36 1.6. MULTIGENOME AND MULTIGENE ANALYSES IN THE APICOMPLEXA 38 1.7. THE OBJECTIVES THIS STUDY WAS TO: 41 Chapter II: Molecular identification ofEimeria species infecting market-age meat in commercial flocks in Ontario 42

2.1. ABSTRACT 42 2.2. INTRODUCTION 42 2.3. MATERIALS AND METHODS 44 2.3.1. Samples 44 2.3.2. Sample Processing and Oocyst Preparation 44 2.3.3. Multiplex PCR Identification of Coccidia 45 2.3.4. Statistical Analysis 46 2.4. RESULTS 46 2.4.1. Histological Observations 46 2.4.2. Prevalence of Infections with spp 46 2.4.3. Multiplex PCR Identifications 47 2.4. DISCUSSION 47 Chapter III: Phylogenetic Position of the Adeleorinid Coccidia (, Apicomplexa, Coccidia, , ) Inferred using 18S rDNA sequences 54 3.1. ABSTRACT 54 3.2. INTRODUCTION 55 3.3. MATERIALS AND METHODS 57 3.3.1. Parasite Material 57 3.3.2. DNA extraction and rDNA PCR Amplification 58 3.3.3. Phylogenetic analyses 60 3.4. RESULTS 62 3.4.1. Parasite Sequences: 62 3.4.2. Phylogenetic Analyses: 62 3.5. DISCUSSION 67 4.1. ABSTRACT 78

ii 4.2. INTRODUCTION 79 4.3.1. Oocysts and DNA extraction 82 4.3.2. PCR reaction parameters 83 4.3.3. Sequence Alignment and Phylogenetic Analysis 84 4.3.3. Species delimitation using mt COI or nu 18S rDNA sequences 87 4.4. RESULTS 88 4.5. DISCUSSION 92 4.5.1. DNA Barcoding for Parasite Species Identification 93 4.5.2. Use of mt COI partial sequences for molecular phylogenetics 96 CHAPTER V: Molecular Phylogenetics of Eimeriid coccidia (Eimeridae, Emeriorina, Apicomplexa, Alveolata): A Multi-gene and Multi-genome approach 101 5.1. ABSTRACT 101 5.2. INTRODUCTION 102 5.3. MATERIALS AND METHODS 104 5.3.1. Sources of Parasites and Parasite DNA 104 5.3.2. DNA Extraction: 105 5.3.3. PCR 105 5.3.4. Phylogenetic Analysis 106 5.3.4.1 Sequencing and Sequence Alignments 106 5.3.4.2. Data Analysis 110 5.4. RESULTS Ill 5.4.1.18S rDNA sequence analysis Ill 5.4.2. Cytochrome c oxidase subunit I sequence analysis 120 5.4.3. Plastid gene analysis 123 5.4.4. Multiple gene and genome consensus tree 126 5.5. DISCUSSION 128 6.0. GENERAL DISCUSSION AND CONCLUSIONS 133 7.0. REFERENCES 141 8.0. APPENDICES 172

111 LIST OF TABLES TABLE 1.1. TAXONOMIC CLASSIFICATION OF TAXA IN THE PHYLUM APICOMPLEXA 8

TABLE 2.1. DETECTION OF EIMERIA SPECIES IN MARKET-AGE MEAT FROM COMMERCIAL BROILER FLOCKS 51

TABLE 3.1. ORGANISMS ANALYZED PHYLOGENETICALLY USING NUCLEAR 18SRDNA SEQUENCES 73

TABLE 4.1. COMPARISON OF PAIRWISE DIFFERENCES (MEAN±STANDARD ERROR) OF 18S NUCLEAR RDNA GENE SEQUENCES AND MITOCHONDRIAL CYTOCHROME OXIDASE C SUBUNIT 1 (COI) SEQUENCES 99

TABLE 4.2. COMPARISON OF THE GENETIC VARIATION WITHIN SPECIES AND BETWEEN CLOSEST SPECIES OF SEVEN ELMERIA SPECIES OF CHICKENS 100

TABLE 5.1. PLASTID AND MITOCHONDRIAL GENE PRIMER SETS 106

IV LIST OF FIGURES

Figure 1.1: Representation of the probable evolutionary relationships among major groups within the Alveolata, based principally on 18S rDNA sequences 17 Figure 1.2: General layout of nuclear genes (18S rDNA; large subunit, 28S; 5.8S and 5S) ribosomal gene in 32 Figure 1.3: Mitochondrial genome structure of Eimeria tenella and falciparum 34 Figure 1.4: Gene organization oftheplastid genome of Eimeria tenella 37 Figure 2.1: Histological appearance of the cecal epithelium of a commercial broiler that had visible macroscopic cecal lesions and tested positive for Eimeria tenella using multiplex PCR 52 Figure 2.2: RAPD-SCAR-based multiplex PCR products obtained when using single Eimeria species as templates 52 Figure 2.3: Typical agarose gel showing separated multiplex PCR products with positive amplification products from individual DNA samples from intestinal contents containing oocysts 53 Figure 3.1: Bayesian phylogenetic analysis using 85 18S rDNA sequences from members of the Apicomplexa including the adeleorinid coccidia 65 Figure 3.2: Ingroup Bayesian phylogenetic analysis using 78 18S rDNA sequences from adeleorinid coccidia 66 Figure 4.1: Phylogenetic trees of coccidia based on Bayesian analyses of nuclear 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences 89 Figure 5.1: Consensus Bayesian tree generated from 223 taxa of 18S rDNA sequences (GTR+I+G) with posterior probabilities of clade support 114 Figure 5.2: Consensus Bayesian tree generated from 101 subset taxa (represented across all three genomes) of 18S rDNA sequences (GTR+I+G) with posterior probabilities of clade support 119 Figure 5.3: Consensus Bayesian tree generated from 101 subset taxa cytochrome oxidase c I sequences (GTR+G) with posterior probabilities of clade support 121 Figure 5.4: Consensus Bayesian tree generated from 85 subset taxa cytochrome oxidase c I sequences (GTR+G) with posterior probabilities of clade support 122 Figure 5.5: Consensus Bayesian tree generated from 23 (GTR+I+G) concatenated available plastid sequences (LSU, SSU, rpoB 1 and rpoB) with posterior probabilities of clade support 124 Figure 5.6: Consensus Bayesian tree generated (GTR+I+G) from 14 subset taxa of concatenated plastid gene sequences (LSU, SSU, rpoBl and rpoB) with their posterior probabilities 125 Figure 5.7: Consensus Bayesian "total evidence" tree generated (GTR+I+G) from 11 taxa of concatenated strict consensus sequences from plastid (pis.) mitochondrial (mt.) and nuclear (nu.) gene sequences with their posterior probabilities 127

V LIST OF APPENDICES

APPENDIX 1. Collection and Infection Data for Market-Age Commercial Broiler Chickens, 2007 172 APPENDIX 2: All newly generated sequences from apicomplexan parasites and their GenBank accession numbers 173

vi LIST OF ABBREVIATIONS

°C Degree Celsius ul microliter AFLP Amplified Fragment Length Polymorphism AT Adenine and Thymine Bgl iill Restriction Endonuclease Bacillus globigii II BLAST Basic Local Alignment Search Tool bp Base pairs CI Confidence Interval COI Cytochrome c Oxidase Subunit I DNA Deoxyribonucleic acid EcoRl : Restriction Endonuclease Escherichia coli Rl EDTA Ethylenediaminetetraacetic acid GC Guanine and Cytosine GTRG General Time Reversible model with Gamma distribution IGS Intergenic Spacer Regions ITS Internal Transcribed Spacer LSU Large Subunit MEGA Molecular Evolutionary Genetics Analysis mins Minutes ML Maximum Likelihood MP Maximum Parsimony mt Mitochondrial ng nanogram NSERC Natural Science and Engineering Research Council nu Nuclear OMAFRA Ontario Ministry of Agriculture, Food and Rural Affairs ORFs Open Reading Frames PAUP Phylogenetic Analysis Using Parsimony PCR Polymerase Chain Reaction RAPD Random Amplified Polymorphic DNA RFLP Restriction Fragment Length Polymorphism RNA Ribonucleic Acid RpoB Ribosomal Polymerase B RpoBl Ribosomal Polymerase Bl SCAR Sequence Characterised Amplified Region SSU Small Subunit TAE Tris-acetate-EDTA V Volt

vn DECLARATION OF WORK

All work reported in this thesis were performed by Joseph D. Ogedengbe with the following exceptions:

Parasites used in Chapter 3 were isolated by John Barta and 18S rDNA were amplified and cloned by Sue Kopko and Shibesh Basak under the supervision of J. Barta.

Joseph Ogedengbe generated consensus sequences from these clones

1 CHAPTER I: INTRODUCTION AND LITERATURE REVIEW

1.1. Introduction

The phylum Apicomplexa contains obligate protistan parasites including some important disease-causing agents that affect humans and their domesticated animals.

Members of the Apicomplexa of medical and/or veterinary importance include

Plasmodium, enteric and tissue coccidia, piroplasms and . Members of the phylum Apicomplexa all belong to the Alveolata (Cavalier-Smith, 1991). The other major taxonomic groups belonging to the Alveolata include the early branching and , most of which are free living organisms. Within the phylum

Apicomplexa, major recognized groups include the gregarines, Cryptosporidia, adeleids, piroplasms, hemosporinids, and the coccidia (toxoplasmids, sarcocystids and the eimeriids). Over the years the classification of these groups within the phylum has been revised repeatedly as more information and better means of accurate descriptions became available. Morphological, biological, biochemical and molecular data have been used to define and redefine groups within the Apicomplexa (Levine and Ivens, 1972; Levine,

1985; 1988; Adl et al., 2005).

One of the main characteristic of all members of the Apicomplexa is the possession of an apical complex at some point in their life cycles. The apical complex consists of , conoid, and cellular inclusions that allow the penetration or the association between the parasite and its host cells. Other cellular inclusions may include plastids and mitochondria in some groups of the Apicomplexa, however; these cellular organelles have not been identified conclusively in other groups. The lack of clearly homologous morphological characters and variability of life cycle stages in the

2 Apicomplexa have led to unreliable classifications, especially within certain groups such as the adeleorinid coccidia (Siddall, 1995). Major revisions of some groups have been necessitated by the fact that representative parasites were obtained from few host species.

In the last two decades, molecular and morphological data have been evaluated in order to study the relationships amongst the members of the Apicomplexa by several researchers (e.g. Barta et al., 1991; Barta, 1989; Cavalier-Smith, 1993; Tenter et al,

2002; Morrison et al, 2004; Morrison, 2008).

Most apicomplexan possess three genomes, namely; the nuclear, mitochondrial and plastid genomes. Cytoplasmic organellar genomes and genes suggest the possibility of generating more broadly based molecular data to confirm or refute proposed phylogenetic hypotheses (Gray, 1993; Wilson and Williamson 1997). Nuclear

18S rDNAs have been widely used in the molecular phylogeny of the Apicomplexa, though its variability has made it difficult to establish positional homology among distantly related sequences in phylogenetic studies (Barta et al., 1997; Li et al., 1997;

Bhoora et al., 2009). The 28S rRNA gene has also been used with some success in resolving species differentiation, and parasite host coevolution in the

(Mugridge et al., 1999). Plastid organelles have been described in some members of the

Apicomplexa and their genes have been used to resolve the phylogeny of Eimeria,

Plasmodium, and members of the Sarcocystidae (Gardener et al., 1994; Zhao et al., 2001;

Zhao and Dusynski, 2001; Obornik et al., 2002; Cai et al., 2003). Few studies have been done on the plastid genes and genome of the Eimeria of food animals however.

Complete mitochondria are not found universally within the phylum Apicomplexa.

However, apicomplexan protists lacking complete mitochondria do have mitochondrion-

3 derived organelles such as mitosomes or hydrogenosomes, as well as chaperonin 60 and heat-shock proteins that were originally encoded by mitochondrial genes (Embley et al.,

2003). The sister group to the Apicomplexa, the dinoflagellates, possess complete mitochondria suggesting that apicomplexan parasites without complete mitochondria likely underwent secondary loss of this organelle.

Mitochondrial gene markers such as the cytochrome oxidases have been used in molecular phylogeny and identification of both apicomplexan and other parasites (Cunha et al., 2009; Alcaide et al., 2009; Ferri et al., 2009) and its possibilities as a DNA barcoding marker was highlighted by Hebert et al. (2003; 2004a). Most studies on mitochondrial genes in the Apicomplexa have utilized taxa within the hemosporinids and

Eimeria (Perkins and Schall, 2002; Cunha et al., 2009; Schwarz et al., 2009). There is therefore a need to explore the mitochondrial genome and genes of further members of the Apicomplexa in order to elucidate phylogenetic hypothesis that may be less clear with other genes.

The purpose of this review is to examine the current literature on members of the

Apicomplexa in the area of diagnosis, classical systematics, current systematic hypotheses, useful phylogenetic markers, and molecular phylogeny in order to learn from it and identify areas that need attention.

1.2. The Phylum Apicomplexa

According to Adl et al. (2005), molecular phylogenetic studies group eukaryotes into six groups, namely; the Opisthokonta, , , ,

Archaeplastida and . Chromalveolata includes the Alveolata (ciliates, dinoflagellates, Apicomplexa), the Stramenopiles (brown , diatoms, many zoosporic

4 fungi, and the opalinids amongst others), with the Haptophyta and . The group is thought to have acquired a plastid through secondary endosymbiosis with an ancestral archaeplastid which became secondarily lost in some with a tertiary reacquisition of plastid in others (Adl, et al 2005). The group Alveolata is characterized by the presence of cortical alveoli beneath the cellular wall Cavalier-Smith (1991). The major groups within the Alveolata include the Dinoflagellates (e.g family , and Dinoflagellata). The phylum Apicomplexa (Levine, 1970) consists of several groups which have at least one stage of the life cycle with flattened subpellicular vesicles and an apical complex consisting of one or more polar rings, rhoptries, micronemes, conoid, and subpellicular . Sexuality, where known, is by syngamy followed by immediate meiosis to produce haploid progeny; asexual reproduction of haploid stages occur by binary , endodyogeny, endopolyogeny and/or schizogony. Locomotion is by gliding, body flexion, longitudinal ridges, and/or flagella.

All members of the phylum Apicomplexa are parasitic except which are free-living (Adl et al., 2005). Apart from the class Colpodellida Cavalier-Smith,

1993, Mehlhorn et al. (1980) suggested that the Apicomplexa can be broadly divided into the Mehlhorn, Peters & Haberkorn 1980 consisting of Hemosporidida

Danilewski 1885 (, , , Plasmodium,) and the

Piroplasmorida Wenyon 1926 (, Cytauxzoori). The Aconoidasida are characterized by the presence of an incomplete apical complex because they lack conoids in most if not all motile stages (note: some hemosporid parasites possess complete conoids in the ookinete stage). They may have motile sexual stages with macrogametes and microgametes forming independently. The , according to Levine (1988)

5 consist of a group in the Apicomplexa that have complete apical complex, including a conoid in all or most asexual motile stages; flagella, where present, are found exclusively in microgametes (male gametes); with the exception of microgametes, motility generally via gliding with possibility of body flexion and undulation of longitudinal pellicular ridges; may be heteroxenous or homoxenous. This group is not monophyletic.

Subdivisions are artificial and subject to future revisions (Adl et al., 2005). The groups within the Conoidasida include Coccidiasina Leuckart 1879 where mature gametes develop intracellularly with numerous microgamete and non-motile zygotes with sporozoites normally formed within sporocysts and oocysts (e.g. Cryptosporidium,

Eimeria, Toxoplasma, , , and ). Some groups within the Conoidasida undergo syzygy (association of pre-gametes [gamonts]) prior to gamete maturation and syngamy. The (Dufour 1828) are characterized by syzygy and with mature gamonts developing extracellularly; syzygy of gamonts generally precedes production of a gametocyst; similar numbers of macrogametes and microgametes maturing from paired gamonts in syzygy within the gametocyst produce few to many oocysts, each of which contain sporozoites. Sporocysts are absent and asexual replication (merogony) is also absent in some species (e.g. Gregarina, Levinea,

Menospora, Nematocystis, Nematopsis, Steinina, and Trichorhynchus, see Adl et al.

2005). Generally, members of the Apicomplexa are parasitic and there is a lot that is unclear in the classification of certain groups that had been erected purely on morphological descriptions and life cycle stages. The apical complex appears to be important at some stage of the parasite in the invasion of the host cells but its function in those parasites that do not invade cells is still unknown (Perkins et al, 2000). Table 1.1

6 shows the classification of the phylum Apicomplexa and genera within major groups according to Levine (1988). TABLE 1.1. TAXONOMIC CLASSIFICATION OF TAXA IN THE PHYLUM APICOMPLEXA ACCORDING TO LEVINE (1988)

PHYLUM APICOMPLEXA Levine, 1970

Class Perkinsasida Levine, 1978 Levine, 1978

Class Conoidasida Levine, 1988 Subclass Gregarinasina Dufour, 1828 Order Archigregarinonda Grasse, 1953 Genera (examples) Selemdioides Levrne, 1971, Exoschizon Hukui, 1939 Order Eugregarinonda Leger, 1900 Suborder Blastogregannonna Chatton and Villeneuve, 1936 Suborder Aseptatorma Chakravarty, 1960 Genera (examples) Monocyshs von Stem, 1848, Lankesteria Mingazzini, 1891 Suborder Septatonna Lankester, 1885 Genera (examples) Gregarina Dufour, 1828, Stylocephalus Ellis, 1912 Order Neogregannonda Grass£, 1953 Genera (examples) Ophriocyshs Schneider, 1883, Schizocystis L6ger, 1900 Subclass Coccidiasina Leuckart, 1879 Order Protococcidionda Kheisin, 1956 Genera (examples) Grelha Levine, 1973, Coelotropha Hennerd, 1963 Order Eucoccidionda Leger and Duboscq, 1910 Suborder Adeleonna Leger, 1911 Family Haemogregannidae Leger, 1911 Genera Danilewsky, 1885, Hepatozoon Miller, 1908 Suborder Eimerionna Ldger, 1911 Family Cryptospondndae Leger, 1911 Genus Cryptosporidium Tyzzer, 1907 Family Eimerudae Minchin, 1903 Genera Eimena Schneider, 1875, Cyclospora Schneider, 1881, L6ger, 1904, Isospora Schneider, 1881 Family Atoxoplasmatidae Levine, 1982 Genus Gamham, 1950 Family Lankesterellidae Noller, 1920 Genus Lankesterella Labbe, 1899 Family Sarcocystidae Poche, 1913 Subfamily Sarcocystmae Poche, 1913 Genera Sarcocystis Lankester, 1882, Frenkeha Biocca, 1968 Subfamily Toxoplasmatinae Biocca, 1957 Genera Toxoplasma Nicolle and Manceaux, 1909, Henry, 1913

Class Aconoidasida Mehlhorn, Peters, and Haberkorn, 1980 Order Haemosponna Danilewsky, 1885 Family Plasmodudae Mesnil, 1903 Genera Plasmodium Marchiafava and Celli, 1885, Dionisi, 1899, Hepatocystis Levaditi and Schoen, 1932 emend Garnham, 1951,/faemoprofeu.sKruse, 1890, Leucocytozoon Sambon, 1908 Order Piroplasmonda Wenyon, 1926 Family Babesndae Poche, 1913 Genus Babesia Starcovrci, 1893 Family Theilerndae du Toit, 1918 Genus Theileria Bettencourt, Franca, and Borges, 1907

8 1.3. Methods of Identification

One of the problems confronted by parasitologists is to identify accurately the species of parasites with which they are dealing, especially when several parasite species can infect the same host and there is the possibility of having several parasites infecting a host at the same time. This is compounded by the morphologically diverse life cycle stages found within the life cycle of a single apicomplexan parasite.

Morphological identification has been the main means of identifying species in the phylum Apicomplexa from early studies (Levine, 1984; 1988). Morphological details in classifying the Apicomplexa generally include size and shape of oocysts, sporocysts, sporozoites and the presence of oocyst and sporocyst inclusions (Perkins et al., 2000).

Other details may include patent and prepatent periods of infection, sporulation times, characteristic lesions and predilection sites (Levine, 1988). The usefulness of morphological details in conjunction with molecular methods have been suggested for identifying and evaluating the systematics of organisms in the phylum Apicomplexa

(Carreno et al 1999; Adl et al 2005; Barta and Thompson, 2006).

Molecular methods that have been used for parasite identification include isoenzyme analyses and unique RAPD-based sequence amplification using PCR (Shirley,

1975; 1997; Tsuji et al, 1997 and Fernandez et al. 2003a). Jenkins et al. (2006),

Cantacessi et al. (2008) and Sun et al. (2009) used internal transcribed spacer regions of ribosomal genes for identifying a number of Eimeria species from poultry litter. A combination of nuclear (ribosomal small subunit and internal transcribed spacer regions) and mitochondrial genes (COI) was used in monitoring the population dynamics of oocysts in flocks under various management conditions (Schwarz et al., 2009).

9 These methods have achieved varying degrees of success in their use as efficient and easily reproducible methods of Eimeria species identification. The shortcoming of using isoenzyme analysis is that oocysts are needed in large numbers from single species/isolates and even then the resulting enzyme profiles sometimes overlap making interpretation difficult; further, isoenzyme analyses require all reference taxa to be run at the same time (greatly increasing the requirement for parasite material that is difficult and expensive to obtain). Random Amplified Polymorphic DNA regions of DNA are useful but the reproducibility of the PCR is dependent on maintaining strictly identical reaction conditions. The use of nuclear genes, especially internal transcribed spacer regions that have multiple gene copies and are not under strong selective pressure, may lead to undesirable paralogous sequences (Lew et al., 2003).

Fernandez et al (2003 a) developed a technique for the simultaneous differentiation of the seven species of Eimeria of chicken in a single PCR reaction. The authors utilized random amplified polymorphic DNA regions that provisionally code for no known proteins in the parasite. The random primers used for RAPDs generated sequences that were used to design specific primers for sequence characterized amplified region

(SCAR)-based markers specific for each Eimeria species. The primers generated were useful in detecting very low numbers of oocysts in a sample (Fernandez et al., 2003a).

However, it is impossible to know if all strains of a particular Eimeria species actually possess the PCR primer sequences targeted in this assay so there is a possibility of underestimating the number of Eimeria species present in a particular sample. A summary of common molecular diagnostic methods for apicomplexan parasites, particularly Eimeria species, was compiled by Morris and Gasser (2006).

10 DNA barcoding of organisms in order to identify them is a relatively recent attempt to aid DNA-based molecular epidemiology and alpha (Besansky et al., 2003).

Mitochondrial cytochrome c oxidase subunit I (mt COI) sequences in conjunction with allozyme analyses have been used in the identification of cryptic species and DNA barcoding of various species including , , and, recently, parasites

(Witt et al, 2000; Hebert et al, 2003; Cunha et al., 2009). According to the Canadian

Barcode Initiative (http://www.dnabarcoding.ca/), "DNA barcodes are generated through the accumulation of random mutations between reproductively isolated groups of organisms. However, with a modest 2% per million year rate of sequence evolution, a

600 bp segment of DNA will, in theory, provide 12 diagnostic nucleotide differences between any two species that have been separated by only one million years" (Hebert et al, 2003). Essentially, DNA barcoding involves determining and comparing the nucleotide sequences of several hundred base pairs from a particular gene (e.g. mt COI) region to differentiate between species (Hebert et al. 2003; Stoeckle and Hebert 2008). It has been suggested as a useful method of studying species diversity in light of current limitation of classical taxonomical markers (Besansky et al, 2003).

1.4. Molecular phylogenetics.

Initially, phylogenetic analyses were based on morphological features exhibited by particular organisms with attendant character inconsistencies and difficulties in clearly classifying or defining species clusters and their intra or inter relatedness because of the paucity of homologous (but variable) morphological characters. In view of these difficulties, molecular taxonomists used molecular data with mathematical (statistical) estimations to arrive at an evolutionary hypothesis for a particular group of organisms.

11 Molecular phylogenetics generally refers to the generation of a hypothesis of relationships among species based on information obtained from one or more molecules

(usually DNA or proteins) and the rigourous, hypothesis-driven analysis of the generated molecular data. Methods used to generate molecular data for phylogenetic study include protein electrophoresis (allozyme and isozymes), immunological methods, protein sequencing, DNA-DNA hybridization, DNA fragment analysis (RFLP, RAPD and

AFLP) and DNA sequencing (see Hillis et al., 1996).

Once molecular data have been obtained, analysis of these data follows. Initially, phenetic methods (based on genetic 'distance' rather than character state analyses) that assumed a more or less constant molecular clock were employed (e.g. Unweighted Pair

Group Method with Arithmetic Mean [UPGMA] and Neighbor Joining [NJ]). These strictly numerical distance methods were replaced by non parametric and non model based methods based on character analysis (e.g. parsimony analysis). Although limited nucleotide substitution models could be employed within the character-based parsimony analysis, more elaborate models of nucleotide substitution (taking into account variation among taxa and among sites within the sequences being analyzed) were analyzed more computationally efficiently using Maximum likelihood and Bayesian methods. In the following paragraphs, the development of ever more sophisticated methods of molecular phylogenetics is described along with the basic principles underlying each method.

The earliest method of molecular data analysis (not phylogenetics) was phenetic analysis based on genetic distance (NJ and UPGMA) that all assume a constant rate of nucleotide evolution or the molecular clock theory (Zuckekandl and Paulin 1962). Fitch and Margoliash (1967) using mt COI were among the first to use genetic distances among

12 sequences based on the rates of mutation for phylogenetic estimates. The authors called their method the 'distance method' because it dealt with mutation distance as described by the authors thus - "The mutation distance between two cytochromes is defined as the minimal number of nucleotides that would need to be altered in order for the gene for one cytochrome to code for the other". Using this method Fitch and Margoliash (1967) introduced the concept of genetic distance and suggested a mathematical basis for dealing with phylogenetic relationships in data containing large numbers of taxa or species.

The accuracy of distance methods can sometimes be enhanced by subjecting the trees to pseudostatistical tests of support such as bootstrapping (Felsenstein, 1985) in order to estimate the relative confidence for the branching order found on the optimal tree(s). Fitch (1971) suggested that in order to better deduce evolutionary relationships; a simple interpretation must be sought in explaining tree topologies generated from a phylogenetic hypothesis. Fitch's idea introduced the concept of parsimony (Hennig,

1966) to molecular phylogenetic analyses, where minimum evolutionary changes are used to explain phylogenetic hypothesis. Both neighbor joining and parsimony are non parametric and non model based methods of data analysis in their most basic forms. The methods have drawbacks in terms of rates of nucleotide substitution within and across operating taxonomic units of phylogenetic reconstructions. Felsenstein (1982; 1983;

1985) introduced the parametric maximum likelihood approach to phylogenetic analysis and suggested bootstrapping as a tool for estimating support (relative confidence) for phylogenetic tree topologies. Holmes (2003) further elaborated on the usefulness of the methods suggested by Felsenstein (1985). Bayesian analysis was introduced by several authors (Mau et al., 1999; Rannala and Yang 1996; Yang and Rannala, 1997) in order to

13 address some of the shortcomings of likelihood methods. Larget and Simon (1999) enumerated the usefulness of Bayesian analysis in multi-sequence analysis, especially as it relates to computational ease over maximum likelihood through the use of Markov

Chain Monte Carlo algorithm.

Most molecular phylogenetic analyses based on parametric evaluations of nucleotide substitution models assumes the absence of a constant molecular clock across sites among taxonomic units. Nucleotide substitution models are used to arrive at the best rates of nucleotide substitutions across taxa and the implications of these changes over time (Sullivan and Joyce, 2005). Model testing has become a necessary preliminary process that subjects DNA or protein datasets to substitution models. Optimization for the best model that fits the data is usually based on the hierarchical likelihood ratio tests.

Posada and Crandall (1998; 2001) proposed the use of the hierarchical likelihood ratio test and the Akaike Information Criterion for model selection in phylogenetic analyses and called it Modeltest. Posada and Buckley (2004) suggested the use of the Akaike

Information Criterion and Bayesian Information Criterion as better tools than the use of only hierarchical likelihood ratio tests. The number of models suggested by Posada and

Crandall (2001) was reduced from 56 to 25 by Nylander (2004) especially for maximum likelihood and Bayesian analsyses and called the test MrModeltest. Recently, Posada

(2008) developed a stand-alone program for model testing, called jModeltest that incorporates more criteria for estimating genetic mutation and nucleotide substitutions in sequences for phylogenetic analyses. The jModeltest utilizes the hierarchical likelihood ratio tests, dynamic likelihood ratio test, and model ranking criterion by the Akaike

Information Criterion and Bayesian Information Criterion or decision-theoretic

14 performance-based approach, Posada (2008). Despite the usefulness of model testing in molecular phylogeny it has become important to assess individual models given the data, to avoid misspecifications and bias due to overparametrization or underparameterizaition as discussed by Lemmon and Moriarty (2004).

Most molecular phylogenetic studies use DNA or protein sequences from organisms in order to obtain molecular information for formulating phylogenetic hypothesis. Avise (1994) gave a summary of useful molecular markers for phylogenetic studies depending on the particular hypothesis to be tested. The sequences (DNA, RNA and proteins) are obtained from homologous genes that are shared among the organisms under consideration and vary sufficiently among the study organisms that sufficient phylogenetically informative data are available generating an evolutionary hypothesis.

Molecular phylogenetic studies have necessitated theories on how genes behave in a population and how these behaviours affect the gene's evolution. Kimura (1968) proposed the neutral theory of evolution in which evolutionary changes are generated primarily by genetic drift and the vast majority of generated mutations are neutral and have no adverse effect on the fitness of the individual organism. Kingman (1982) proposed the coalescent theory of evolution which is a retrospective approach to population genetics. The coalescent theory forms a gene genealogy by tracing the ancestry of particular genes and establishing ancestral lineages. Griffths and Tavares

(1994) and Khuner et al. (1995) introduced the Markov-Chain Monte Carlo to population genetics. The aim of most molecular phylogenetic analyses is to construct a species tree on the basis of gene trees generated from gene sequence analysis and appropriate models of nucleotide substitutions (see discussion above). To be a useful marker, a gene must be

15 common among the studied organisms and must be heritable, conserved, and variable

enough to provide phylogenetically informative characters for analysis. For such studies,

it is best to sequence orthologous genes which arise by vertical descent from a single

ancestral gene and not by duplication as in paralogous genes (Hillis et al., 1996). The

general conceptual basis of terms relating to homology and relationships between

different types of homologous genes and character estimation in molecular phylogeny

was summarized by Fitch (2000).

In summary, current molecular phylogenetic analyses combine generation of an a priori, dataset-specific nucleotide substitution model(s) that is then used in a character-

based tree construction method (usually based on parsimony, maximum likelihood or

Bayesian computations). An estimate of the 'robustness' of the resulting evolutionary

hypothesis can then be calculated using the data-resampling bootstrapping method

(Felsenstein, 1985) for maximum likelihood and parsimony analyses or posterior

probability for Bayesian analyses.

1.4.1. Molecular Phylogeny of the Apicomplexa (Levine, 1970)

The phylum Apicomplexa has received considerable attention because of the

importance of its members as parasites but the molecular systematics of the group is still

a topic of considerable research. The molecular phylogeny of apicomplexan parasites has been primarily based on the nuclear genes, especially the 18S rDNA (e.g. Barta et al.,

1997; Barta et al.2001; Morrison et al., 2004, Morrison, 2008; 2009).

16 Gliotcs

Di no flagellate;

Colpodcllids

Cryptosporidium species

Grcjarinct

> Adclcid Cotcidia o' o 5 Pi ooplasms N * 3 O N 2. Plasmodium O species X D D D O

5arcocystina« and T»xoplasmatin«ie ("Tissue Coecidia")

Eimeriid Coccidio

Figure 1.1. Representation of the probable evolutionary relationships among major groups within the Alveolata, based principally on 18S rDNA sequences. Only the branching order among the taxa is shown. Uncertainty regarding the monophyly of several of these widely recognized groups is represented as dotted horizontal lines. The monophyly of the Apicomplexa is well supported, with many of the traditional groups of apicomplexan parasites, such as the malarial parasites or tissue coccidia, shown to be natural (monophyletic) groupings. (Reprinted from Barta and Thompson, 2006, with permission).

17 The phylum Apicomplexa belongs to a large group of protists, the Alveolata, that

possess homologous subpellicular vesicles (cortical alveolae); this synapomorphic feature

has been supported in a large number of molecular phylogenetic studies (e.g. Fig. 1.1).

The ciliates branched early among the protists followed by the dinoflagellates.

After the clade are the colpodellids followed by the Apicomplexa sensu

stricto (e.g. archigregarines, gregarines, Cryptosporidia, hemosporids, Aggregatidae,

piroplasms and coccidia (Sarcocystidae and among others). The phylogeny of

taxa within the Apicomplexa is poorly understood due to paucity of robust studies

incorporating extensive taxon sampling, sufficient sequence length and divergence,

alignment stressing positional homology, appropriate genetic markers and phylogenetic

analyses with robust analytical procedures that take into account the nucleotide

substitution rates within and across taxa (Morrison, 2008). Within the major groups of

Apicomplexa such as the Sarcocystidae and Eimeriidae, phylogenetic analyses and

molecular identification studies have shown the difficulties of species delineations,

especially within the genera , , Toxoplasma and

(Ellis et al., 1999; Mugridge et al., 1999) and the paraphyly of the family Eimeriidae (see

Morrison et al., 2004). Levine (1988) estimated that there were over 300 genera and

about 4800 named species in the phylum Apicomplexa at that time, but suggested that there were in reality far more than this estimate. A tiny fraction of about 6000 species have been described from an estimated 1.2-10 million extant species (Adl et al., 2007;

Morrison, 2009). The diversity of the Apicomplexa has been the source of constant

challenge in view of the inherent bias towards disease agents that are particularly

important to man. The most studied species of apicomplexan parasites include some

18 hemosporinids, piroplasms and coccidia, because they include important genera that have both medical and veterinary medical importance. Molecular phylogenies have therefore been based on medical exigencies rather than genuine phylogenetic inquiry (Morrison,

2009). With this limitation clearly recognized, it is necessary to identify and examine early branching apicomplexan taxa for the proper rooting of phylogenetic trees to satisfactorily identify the relationships within and between apicomplexan clades

(Morrison, 2008; 2009). The objective of most molecular phylogenetic studies is to confirm or refute classical taxonomic hypotheses relating to particular organisms. Based on the limited data available to date, the earliest diverging taxa within the Myzozoa are the dinoflagellates followed by the colpodellids and then, within the phylum

Apicomplexa, the archigregarines and gregarines; all of these latter groups have had fewer taxa sampled for phylogenetic studies. Leander et al. (2003) noted that basing the molecular phylogeny of the Apicomplexa on a few representative taxa might result in spurious conclusions. It was further observed that the variation of morphological features within the Apicomplexa such as the apical complex in Perkinsida Levine, 1978 and

Colpodella Cienkowski, 1865, and the lack of comparable life cycle phases such as merogony, gametogony and sporogony in all genera makes extensive taxon sampling imperative and makes it difficult to reach a consensus as to which characters to use in delineating the Apicomplexa (Gajadhar et al., 1991; Leander et al., 2003). Members of the Perkinsida are marine apicomplexan parasites infecting marine molluscs. One of the first descriptions of a member of Perkinsida (Dermocystidium marinum) was by Mackin et al. (1950). Subsequent descriptions and classification by Mackin, (1951); Mackin and

Ray, (1966) led to the conclusion of the authors that the newly described parasite of

19 is a . Perkins and Menzel (1967) described Dermocytidium marinum as belonging to the Saprolegniales, an order of fungi, based on its ultrastructure and life stages. However, these conclusions were revised based on the descriptions of Perkins

(1976) of Dermocystidium marinum in the American , Crassostrea virginica, when it was accepted as a and a legitimate member of the Apicomplexa because of the presence of an apical complex (Levine, 1978). Molecular phylogenetic analysis including a Perkinsus species demonstrated that these parasites were more closely related to the dinoflagellates and other members of the Alveolata (Goggin and Barker, 1993) rather than to fungi or flagellates as previously thought. The phylogenetic position of the class Perkinsida has been controversial and Reece et al. (1997) considered the 18S rDNA; a non protein-coding gene with hypervariable regions that make establishing homology difficult, a less than perfect molecular genetic marker. In contrast, Siddall et al. (1997) supported the use of nu SSU rDNA sequences and was of the opinion that a protein-coding gene, such as actin, might be susceptible to codon bias. The perkinsids were found to be more closely related to the dinoflagellates than to other members of the

Apicomplexa using an actin gene as a molecular marker (Reece et al., 1997; Siddall et al.,

1997; Siddall et al., 2001). In order to reduce the disadvantages of using two dissimilar genes, (18S rDNA and tubulin) Siddall et al. (1997) used a total evidence phylogenetic approach by combining morphological, 18S rDNA, and actin nucleotide data to investigate the relationships among Perkinsus spp. and other members of the Alveolata.

The study concluded that Perkinsus spp. were more closely related to the dinoflagellates than the Apicomplexa despite the spurious association of Plasmodium and

Cryptosporidium as a clade and the grouping of the Apicomplexa with the ciliates. The

20 study by Reece et al. (1997) using the actin gene alone gave a better resolution of the groups within the Apicomplexa even though they still came to the same conclusion that

Perkinsus was more closely related to the dinoflagellates. In these studies the number of taxa used for each analysis was limited and there were few early branching members of the Apicomplexa such as the archigregarines and gregarines included in these analyses; these early diverging apicomplexan groups could be useful in showing the relationship of

Perkinsus species to other members of the Apicomplexa. Generally, Perkinsus species and colpodellids have symplesiomorphic features that make them useful early branching taxa in apicomplexan phylogeny (Siddall et al, 2001; Kuvardina et al., 2002) even though the colpodellids regularly form a sister group relationship with the remaining apicomplexan parasites rather than Perkinsus species (Kuvardina et al., 2002).

Gregarines are thought to be extant representatives of the earliest lineage of apicomplexan parasites in the coelom, intestine or reproductive organs of marine and terrestrial with many, especially eugregarines, infecting insects (Leander,

2008). Gregarines are classified according to habitat, host range and trophozoite morphology into Archigregarines, Eugregarines and Neogregarines (Leander 2008).

Archigregarines are found in marine habitats, possess intestinal trophozoites that are similar in morphology to infective sporozoites and are inferred to be most representative of the ancestral gregarine (Levine, 1976; Leander, 2008). Eugregarines are found in marine, freshwater and terrestrial habitats and possess large trophozoites that are significantly different in morphology and behavior from the sporozoites. Intestinal eugregarines are separated into septate and aseptate (mostly marine) gregarines depending on whether the trophozoite is superficially divided by a transverse septum.

21 Urosporidians are aseptate eugregarines that infect the coelomic spaces of marine hosts.

Monocystids are aseptate eugregarines that infect the seminal vesicles of terrestrial annelids and are closely related phylogenetically to neogregarines.

Neogregarines are found in terrestrial hosts (e.g. insects), have reduced trophozoites and tend to infect tissues other than the intestines (Leander, 2008). Phylogenetic analysis shows that the gregarines form a close association with Cryptosporidium and this has led many authors to conclude that the traditional belief of Cryptosporidium belonging to the coccidia needs to be revised based on both morphological and molecular data (Carreno et al., 1999; Leander et al, 2003; Barta and Thompson, 2006). Molecular and morphological studies of gregarines may ultimately shed light on the advent of intracellular and host parasite adaptation in marine and terrestrial habitats and also lead to a better understanding of the closest relative of Cryptosporidium species amongst the gregarines (Leander, 2008). Another group within the Apicomplexa that are found in invertebrates is the family Aggregatidae. Species ofAggregata Frenzel 1885 are poorly resolved in small subunit (ssu) rDNA sequence-based phylogenetic reconstructions. Morphologically, they are related to the coccidia by having oocysts with a variable number of sporocysts and sporozoites. Excystation is however through a longitudinal suture on the sporocyst wall. Though appear within the Adeleid clade in some phylogenetic studies, they do not exhibit syzygy, which is decidedly a character most associated with the adeleid coccidia (Kopecna et al., 2006). The phylogeny of the Aggregata is further complicated by the fact they have a low GC content, leading to highly divergent sequences, difficulty in establishing positional

22 homology during sequence alignment and potentially misleading conclusions (Kopecna et al, 2006).

Of all the coccidia within Apicomplexa, the Adeleorinid coccidia are probably the most poorly understood phylogenetically. The haemogregarine genera are

Haemogregarina, Danilewsky 1885; Karyolosus Labbe, 1894; Hepatozoon Miller, 1908;

Cyrilia Lainson 1981; Petit, Landau, Baccam and Lainson 1990 ;

Siddall 1995; Labbe 1894 and Babesiosoma Jackowski and Nigrelli 1956.

The systematics of the adeleorinid coccidia are for the most part based on morphological details and the key feature of syzygy between gametes before their fusion. Probably the most poorly understood groups within the Adeleorinid coccidia are the

Haemogregarinidae Neveu-Lamaire 1901 and Hepatozoidea Wenyon 1926. The life histories of most haemogregarines are not known, especially the definitive hosts and mode of infection. This has led to the lumping of many described parasites into the genus Haemogregarina sensu lato, where over 300 species have been named, usually from only the blood forms (gamonts) of the parasite (Barta and Desser, 1984; 1989;

Siddall, 1995). Because of the biological diversity within the family Haemogregarinidae it was suggested that these parasites be separated into distinct families (e.g. Barta 1989;

Smith and Desser 1997). On this basis, Hepatozoon belongs to Hepatozoidae,

Karyolosus and Hemolivia belong to while Haemogregarina and belong to the Hemogregarinidae. A redefinition of affinities within the group has led to the erection of the genus Desseria (Haemogregarinidae) to include members found in fish

(Siddall 1995; Smith and Desser 1997). The phylogeny of haemogregarines has mostly been based on morphological data, with little corroborating molecular data, except for a

23 few studies that attempted identifying species of Hepatozoon using molecular methods

and 18S rDNA sequences in studying their phylogeny (Mathew et al., 2000; Baneth et al.,

2002; Jakes et al., 2003). Amongst the Hepatozoidae, which are probably the most

specious of the Adeleorinids, there has been confusion in identifying Hepatozoon species

occurring in canines from different biogeographical zones. Hepatozoon canis and H.

americanum found in the Old and New Worlds respectively were initially thought to be

strains of the same species but have now been found to be two distinct species (Baneth et

al., 2002). Members of the genus Hepatozoon appear to be paraphyletic (did not contain

all members descended from a common ancestor in a single clade) in most phylogenetic

studies and utilize terrestrial invertebrates as definitive hosts (see Barta, 1989; Jakes et

al., 2003). Other members of the Adeleorinid coccidia, such as spp., are found

infecting mostly insects and marine invertebrates. Adelina grylli, Adelina dimidiata and

Adelina bambarooniae are found in insects. In phylogenetic studies of the adeleorinid

coccidia, the adeleids tend to form sister groups with the genus Hepatozoon while the

Aggregate appear to form a sister clade with the Adelina/Hepatozoon clade despite the

absence of syzygy during gametogony (Kopecna et al., 2006). The above authors

observed that the rather unusual nucleotide composition of the Aggregata may be responsible for the highly divergent 18S rDNA and subsequent branching and random positions of the group both within and outside of the adeleorinid coccidia. Members of the family Dactylosmatidae, which includes Dactylosoma and Babesiosoma species, predominantly infect anurans and are transmitted by glossophoniid leeches. Based on morphological and life history details, the Dactylosomatidae appear to group with the

other adeleid coccidia and piroplasms but not the eimeriids or haemosporinids and have

24 been suggested as occupying an intermediate position between the Karyolysidae and piroplasms thus providing the necessary link between the piroplasms and the rest of the

Apicomplexa (Barta, 1989; Barta and Desser, 1989).

According to Peirce, (2000), the genus Babesia consists of 112 named species with about 98 occurring in while the genus Theileria consists of 39 species of which most are found in livestock in and transmitted by ixodid ticks. The distribution of these heteroxenous parasites follows the pattern of distribution of their definitive hosts

(Peirce, 2000). Members of the are described based on the forms found in erythrocytes and the type of host parasitized. Though members of this group could be found in animals as diverse as mammals, birds and , they are found mainly in mammals where they can cause considerable economic loss (Peirce, 2000). In the genus

Babesia, molecular phylogenetic analysis using 18S rDNA sequences has shown that

Babesia bigemina and B. bovis are monophyletic while B. equi and B. rhodaini form a separate clade with the genus Theileria (Ellis et al., 1992). Allsopp et al. (2006) noting the paraphyly of the Babesia genera suggested the reassignment of certain members of the genus Babesia into separate clades, based on results obtained from molecular phylogenetic analyses using 18S rDNA sequences. Barta (1989) suggested that the piroplasms arose from a common ancestor with the adeleid coccidia related to the genus

Babesiosoma. In phylogenetic studies, piroplasms are found to be sister groups to members of the adeleid coccidia and not the haemosporinids as previously believed

(Barta, 1989).

The classification of Cryptosporidium species within the Apicomplexa has traditionally been with the other coccidia based largely on morphology and life-cycle

25 details (Levine, 1988). Some recent studies have suggested that Cryptosporidium species

appear unique among the coccidia, forming sister groups with the gregarines to the

exclusion of the other coccidia within the Apicomplexa (Carreno et al., 1999; Barta and

Thompson, 2006 - see Fig. 1.1). In a separate study, using both 18S rDNA and P-tubulin

sequences it was suggested that though the sister group relationship between

Cryptosporidiidae and the gregarines appear to hold true, the statistical support was weak

(Leander et al., 2003). The difference in phylogenetic lineage between gregarines and

Cryptosporidiidae Leger 1911, and their early divergence from the rest of the

Apicomplexa, namely; haemosporinids, coccidia and piroplasms was however not in

dispute according to Leander et al. (2003). Barta and Thompson (2006) however

suggested that the sole genus of the family Cryptosporidiidae are not unusual members of

the coccidia but are a group related to the gregarines, based on both morphological and

biological details that have taxonomic implications. Recent shotgun sequencing of the

genome of the gregarine Ascogregarina taiwanensis, a parasite of larvae, has

confirmed the close association between gregarines and Cryptosporidia (Templeton et al.,

2010).

The hemosporinids, which consist mainly of species in the genera Plasmodium,

Hepatocystis, Leucocytozoon and Haemoproteus, form a sister group with the piroplasms

and the family Hepatozoidea (Carreno et al., 1997; Barta, 1989). It was also suggested by Carreno et al. (1997) that the phylogeny of Plasmodium reflects parasite and vector

coevolution, and adaptations to terrestrial intermediate hosts are probably not phylogenetically informative. Previous systematic classification of the group was based

on morphology and description of life cycle stages (Levine, 1988). Siddall and Barta

26 (1992) suggested that molecular data may be necessary in order to address issues with classifications within the family and other blood parasites, due to the unreliability and difficulty of obtaining homologous and sufficiently diverse morphological and life cycle details. Apart from the 18S rDNA sequences, other molecular markers for phylogenetic studies that have been used to study the phylogeny of

Plasmodium include sequences generated from the mitochondrial genome (e.g. cytochrome oxidase b, Yotoko and Elisei, 2006), plastid genome (e.g. caseinolystic protease C) and nuclear genome such as surface circumsporozoite proteins (McCutchan et al., 1996) or merozoite surface proteins (Tanabe et al., 2007). Amongst the molecular markers available, mitochondrial cytochrome oxidase b appears to have been most extensively used for the molecular phylogenetics of the family Plasmodiidae (Di Fiore et al., 2009). Perkins and Schall, (2002) showed that parasites infecting mammalian intermediate hosts appear to be paraphyletic with Plasmodium reichnowi of being closest to P. falciparum in man. Using the genus Leucocytozoon as outgroup

Perkins and Schall (2002) observed that the Plasmodium and Hepatocystis species of mammalian hosts on one hand and Plasmodium and Haemoproteus of birds and lizards on the other formed distinct clades in analyses using cytochrome oxidase b sequences.

The authors further suggested that the complexity of malarial phylogeny lies in the relationships within the species that affect other than mammals. The estimated time of divergence between and human Plasmodium (P. falciparum and P. reichenowi respectively) was found to coincide with the divergence of the two vertebrate species, leading to the suggestions that they probably share a common ancestral lineage (Escalante and Ayala, 1994; Perkins and Schall 2002). Yotoko & Elisei

27 (2006) while reconstructing the most-likely ancestral hosts at each internal node of

Plasmodium using a cytochrome b gene found that reptilian hosts were probably the common ancestral vertebrate host for the ancestral parasite that ultimately diverged into extant Plasmodium, Haemoproteus and Hepatocystis species. This implies that based on the current distribution of malarial parasites there could have been at least four intermediate host shifts between and within vertebrate classes involving reptiles, mammals, and birds, even though from an evolutionary standpoint only definitive hosts matter in the divergence of the parasites (see Carreno et al., 1997).

The other important members of the Apicomplexa are the coccidia which comprises both tissue and enteric forms. According to Barta et al. (2001) the coccidia clearly group into two well-supported monophyletic clades (each composed of members with shared characters from a recent common ancestor), namely: the eimeriid coccidia including

Eimeria, Caryospora, Lankesterella and Cyclospora species as well as avian Isospora species; and the isosporoid coccidia that include species in the genera Toxoplasma,

Neospora, , Hammondia and Sarcocystis as well as mammalian Cystoisospora

(formerly Isospora) species. Though these groups of parasites are broadly divided into monoxenous and heteroxenous coccidia, both heteroxeny and monoxeny occur within each broad group (Barta et al., 2001). The need for reclassification and reassessment of the phylogeny of the coccidia is based on the fact that most of the previous studies on systematics have been based on morphology and life cycle details (Levine, 1970; 1988;

Cox, 1994). Tenter et al. (2002) suggested the need to classify the coccidia based on both morphological and molecular data. The two subfamilies within the Sarcocystidae are

Sarcocystinae and Toxoplasmatinae (Levine, 1988). Several authors have noted the

28 phylogenetic (based on morphological and molecular data) uncertainties within the

Sarcocystidae (Ellis et al., 1999; Dubey et al., 2002; Modry et al, 2001; Monteiro et al.,

2007). The Sarcocystinae were first thought to comprise of two genera; Sarcocystis and

Frenkelia, but these are now thought to be synonyms (Votypka et al., 1998; Mugridge et al, 1999). However, Jenkins et al. (1999) suggested that the genus Sarcocystis is paraphyletic from analysis based on nuclear ssu rDNA sequences. Within the

Toxoplasmatinae, the position of the genus Neospora and its relationship to other members of the group had been problematic and debated (Barta et al., 2001). Molecular phylogenetic studies of the family Sarcocystidae using large subunit ribosomal DNA sequences showed that is more closely related to Hammondia heydorni species than which was more closely related to H. hammondi (Ellis et al, 1999; Mugridge et al., 1999). Carreno and Barta (1999) suggested the genus Isospora might be polyphyletic. In a revision of the status of some members of the Sarcocystidae, especially regarding the morphology and taxonomy of

Isospora species infecting mammals, Barta et al. (2005) suggested that Isospora species infecting mammals having no that are disporocystic with four sporozoites in each sporocyst belong to the genus Cystoisospora Frenkel 1977, and those that have a

Stieda body as Isospora species. Carreno et al. (1999) had earlier shown through molecular evidence (18S rDNA sequences) that the genus Isospora that do not have

Stieda body should be classified with the Sarcocystidae. The redefinition of Isospora species became necessary in order to address the phylogenetic confusion and morphological dissimilarities of the group that had included members infecting birds and mammals (Carreno and Barta, 1999; Barta et al, 2005).

29 Morphological and molecular phylogenetic characterizations have proven that one or more of the eimeriid genera are paraphyletic (Barta et al., 2001; Tenter et al, 2002;

Morrison et al., 2004). While most members of the eimeriids are homoxenous, some genera such as Caryospora, and Lainsonia, have more than one host and their environmentally resistant oocysts, when formed, possess variable numbers of sporocysts with Stieda bodies that enclose sporozoites. Molecular data appears to have clarified confusing associations that were based on morphological and life cycle details in recent years (Modry et al., 2001; Jirku et al., 2009). In a study to place Eimeria ranae from within a phylogenetic and taxonomic context using ssu rDNA and morphological attributes such as possession of Stieda bodies or valvular sutures, Jirku et al. (2009) found that Eimeria spp. from homoeothermic animals formed a derived monophyletic clade while those from poikilotherms, including E. arnyi, were early branching. Some Eimeria species from reptiles such as E. tropidura and that had sporocysts with valvular sutures rather than Stieda bodies formed a separate sister clade to all coccidia that had Stieda bodies in their sporocysts. A related coccidian parasite, Isospora lieberkhuni (=Hyaloklossia lieberkuehni (Labbe, 1894) Laveran and Mesnil, 1902) from

Rana esculenta, was found to cluster with members of the Sarcocystidae and it too lacked a Stieda body (Modry et al., 2001). Although most commonly reported from mammals, members of the paraphyletic (Morrison et al., 2004) genus Eimeria have been described from lizards inhabiting the Galapagos Islands. Eimeria tropidura; E. galapagoensis and

E. albemariensis as well as Isospora insularius were described from oocysts found in the lizards Tropidurus delanonis and T. albemariensis (see Aquino-Shuster et al., 1990;

Couch et al., 1996). Interestingly, the Eimeria species did not have a Stieda or substieda

30 bodies in their sporocysts but the Isospora sp. oocysts isolated in the same study possessed Stieda bodies. From these studies, it would appear that a key morphological characteristic of members of the Eimeriidae (possession of Stieda bodies) is absent in at least some Eimeria species supporting the conclusion that the genus Eimeria, like the genus Haemogregarina, has become a paraphyletic repository for a wide variety of unrelated, but superficially morphologically similar, parasites. Some members of the family Eimeriidae affecting poikilotherms do have unique morphological features that are not shared with the other Eimeria species affecting other vertebrates; when better understood, these differences are likely to be addressed taxonomically through the erection of new genera.

1.5. Molecular markers and genomes used in Apicomplexan phylogeny.

1.5.1. Nuclear small subunit gene (18S rRNA gene)

The most commonly used genome and gene for molecular phylogeny of apicomplexans are the nuclear genome and, the nuclear 18S rDNA. One of the earliest studies of the gene was by McCutchan et al. (1988) who developed specific oligonucleotides for the identification of the different ribosomal genes in Plasmodium.

Figure 1.2 illustrates the general layout of the nuclear small subunit in relation to other genes with which it is associated. The 18S rDNA exists in multiple copies in tandem repeats that include internal transcribed spacer regions (ITS), intergenic spacer regions

(IGS), 5.8S, 5S, and the large subunit gene region (28S).

31 major rRNA transcript 5S RNA gene -, A

InterneJ Intergenic transcribed spacer (IGS) spacer (ITS) regions regions

1 rDIJ A repeat unit

EccRI EcoRI Bdll IGS1 IGS2

SSU(18S)RNA 5.8S LSU (25-28S) RNA 5S RNA RNA

Figure 1.2: The general layout of nuclear genes (18S rDNA; large subunit, 25-28S; 5.8S and 5S) ribosomal gene in eukaryotes. (From:http://www bioloqv.duke.edu/funai/mvcolab/primers htm)

Over the years, several primer sets have been developed to amplify the small subunit region (-1800 base pairs) of the nuclear genome (Elwood et al., 1985; Medlin et al., 1988; White et al., 1990; Wozniak et al., 1994). In order to study the deeper relationships amongst the Apicomplexa it is important to sequence the conserved regions within the 18S rDNA. Other regions within the rDNA repeat units such as the ITS and

IGS regions that are under less evolutionary pressure vary more in relation to the rest of the gene and might not be useful in studying ancient phylogenetic relationships (Field et al., 1988; Barta et al, 1997). Several molecular phylogenetic hypotheses have been based on molecular information contained in the small subunit ribosomal DNA of the

Apicomplexa and such information have been the basis of revisions and suggestions for understanding the molecular phylogeny of the group, in relation to classical taxonomy

(e.g. Cavalier-Smith, 1993; Carreno et al., 1999; Carreno and Barta, 1999; Tenter et al.,

2002; Morrison, 2004, 2008; 2009). The use of the 18S rDNAsequence in phylogenetic studies though common, has some drawbacks. Barta (1997; 2001) enumerated the

32 difficulties that might arise in establishing both positional and gene homology using 18S rDNA sequences. In parasites such as Plasmodium for example, there are three different types of 18S rDNA expressed during the life cycle of the parasite that differ in primary structures (Li et al., 1997). Carranza et al., (1996) reported two types of 18S rDNA in a metazoan {Dugesia meditaranea). Barta (2001) argued that variation in the number of copies and the variability of evolutionary pressure on different regions of the gene within the Apicomplexa might result in difficulties establishing both sequence and positional homology especially when based on primary sequence alignments. Barta (1997) further suggested the use of a staggered alignment procedure to increase positional homology in aligned 18S rDNA sequences.

The internal transcribed spacer regions (ITS) have been useful as diagnostic markers of members of the Apicomplexa, especially in the epidemiology of important members such as the genus Eimeria (Schnitzler et al, 1998; Lew et al., 2003; Jenkins et al., 2006; Haug et al., 2007; Schwarz et al., 2009; Sun et al., 2009). The ITS and nuclear large subunits have been used with some success to study the molecular phylogeny of members of the Sarcocystidae (Ellis et al., 1999; Mugridge et al., 1999; Monteneiro et al.,

2007) but appear unable to resolve deep phylogenetic relationships amongst mammalian

Eimeria species when compared to the 18S rDNA sequences (Zhao and Duszynski,

2001).

1.5.2. Mitochondrial genome

Mitochondria are cellular organelles that are thought to have been secondarily acquired in the course of evolution. The first eukaryotes to acquire mitochondria appear to have symbiotically incorporated photosynthetic purple bacterium in order to increase

33 their energy efficiency and utilization (see Cavalier-Smith, 2006). In the Apicomplexa, most members do have mitochondria and "mitochondria-like organelles". The theory (Cavalier-Smith, 1983) was proposed to classify all protists without mitochondria as primitive and early branching groups before the acquisition of mitochondrial photosynthetic bacterium. But the fact that the main group of organisms under this classification, Trichomonas, Giardia, Entamoeba and microsporidia, do appear to have organelles and genes (hydrogenosome, mitosomes, heat shock proteins and chaperonin

60) that perform similar functions as mitochondria, and the clustering of Entamoeba and microsporidia with mitochondrial protists and fungi led to the abandonment of the concept of Archezoa (Burri et al, 2006; Keeling, 2007; Van der Giezen, 2009).

The mitochondrial genome of most animals contains at least 37 genes; two for ribosomal

RNAs, thirteen for proteins and twenty-two for transfer RNAs (Boore, 1999).

Apicomplexan parasites that do possess mitochondria have a substantially reduced mitochondrial genome (Fig. 1.3).

A LF LG 10 SO 13 SA cob C0zl IDOL CWt3 iULi H EJ u m in LB 3 SB LE SE LC SF LD 8 LA B SE LF 2 3 SF DDLE l c&xl tub m Mil ™r Dn 1 LCLGSB 10 SA LB LA SD LE S I 13 LD

—i— —I— 1,0 —!— 3,0 4.0 50 6.0 (kh) 20 Figure 1.3. Mitochondrial genome structure of Eimeria tenella (A) and (B). Genes shown above the bold line in each genome have predicted transcriptional directions from left to right; and those below, from right to left. Because the 6.2 kb element of E. tenella mt genome is tandemly repeated, both termini are arbitrary. White boxes indicate protein-coding genes (coxl [=COI], cox3 and cob); fragments of LSU (LA-LG, 1, 2, 3, 10 and 13) and SSU (SA, SB, SD-SF, 8 and 9) rRNA genes are shown by dark and light gray boxes, respectively. (From Hikosakaetal., 2010).

34 Mitochondrial genes have received some attention in recent times as possible genetic markers in phylogenetic studies (Avise, 1994). Mitochondrial genes have been used in the phylogenetic studies of the Apicomplexa, especially as alternatives or in conjunction with nuclear genes (Rathore et al., 2001; Perkins et al., 2007; Perkins, 2008;

Schwarz et al., 2009). The mitochondrial genes used in molecular phylogenetic studies or molecular identification of members of the Apicomplexa were protein coding genes, such as COI) cytochrome oxidase b, (Perkins et al., 2007; Schwarz et al, 2009). For

Plasmodium spp. at least, the mitochondrion has been shown to be inherited uniparentally from macrogametes (Wilson and Williamson, 1997); uniparental inheritance of mitochondrial genes theoretically lacking recombination events is an advantage in studying speciation and phylogeny that is not shared by nuclear encoded genes. The usefulness of mitochondrial cytochrome oxidase in identification and investigation of phylogenetic relationships have been studied in some parasite groups (Pages et al., 2009;

Alcaide et al., 2009; Huang et al., 2009). Martinsen et al. (2008) used mitochondrial COI sequences in addition to plastid and nuclear genes to study the evolutionary relationships and events leading to host switching and diversification in Plasmodium species of birds, mammals and squamate reptiles. Cunha et al. (2009) have also used cytochrome oxidase c genes (COIII and COI) in the molecular epidemiology of Plasmodium by developing highly sensitive (100%) and specific (88%) PCR-based methods for the diagnosis of P. vivax and P. falciparum. Other members of the Apicomplexa have not been studied to the same extent as Plasmodium using mitochondrial genes, primarily for lack of appropriate primers or, in the case of Cryptosporidium species, the complete loss of a mitochondrial genome.

35 1.5.3. Plastid genome

Plastids are thought to be of green algal origin (Kohler et al., 1997; Wilson, 2002).

They were first studied in the genera Plasmodium, Toxoplasma, Eimeria and Theileria in the phylum Apicomplexa (Wilson et al, 1996; McFadden et al., 1996; Roos et al., 1999;

Cai et al., 2003; Wilson, 2005). The importance of the evolutionary origins of parasites

such as Plasmodium and other apicomplexans is related to the evolution and acquisition

of the plastid genome (Kalanon and McFadden, 2010). , an alveolate, is thought to have secondarily acquired photosynthetic plastids and is the closest photosynthetic relative of the apicomplexans (Moore et al., 2008). The presence of plastids or plastid genes has not been demonstrated in all members of the Apicomplexa

(Abrahamsen et al., 2004; Toso and Omoto, 2007; Templeton et al., 2010). The plastids

as unique organelles of some members of the Apicomplexa have been explored as possible targets of drug-based control measures because of their unique fatty-acid

synthesis pathways (Wilson, 2005). According to Lang-Unash (1998) the widespread occurrence of plastids in apicomplexan species and coevolutionary relationship between rDNA located in their nuclear and plastid genomes suggests a single ancient lineage for the organelles.

Figure 1.4 illustrates the general gene organization of the plastid genome for

Eimeria tenella, a circular -34 kb genome consisting of several open reading frames

(ORFs), ribosomal polymerases, tuf A, small (SSU) and large (LSU) ribosomal subunit genes (Cai et al., 2003).

36 Figure 1.4: Gene organization of the plastid genome of Eimeria tenella. (From Cai et al., 2003)

Apart from establishing the origin of plastid genes, workers have used the sequences generated from these genes in the molecular phylogenetics of members of the

Apicomplexa. Obornik et al. (2002) used plastid ssu rDNA sequences to investigate the molecular phylogenetics of coccidia. The coccidia and the hemosporinids were clearly split into separate clades with Hyaloklossia lieberkhuni forming a clade with members of the Sarcocystidae (Obornik et al., 2002). Zhao and Duszynski (2001) suggested that plastid genes are useful in the resolution of close and distant relationships within mammalian Eimeria lineages because of their higher nucleotide substitution rates and reasonable sequence conservation compared with nuclear small subunit genes. Lau et al.

37 (2009) using plastid genes and expanded taxa confirmed the likely origin of plastids and its implication in current sequence-based phylogenetic hypotheses. The caseinolytic protease gene C (clpC) in conjunction with other organellar genes appears to be a useful gene in resolving the phylogenetic relationships within the hemosporidians, especially host-parasite cospeciation (Hughes and Verra, 2010) and the resolution of terminal and internal branching within the group (Outlaw and Ricklef, 2010).

1.6. Multigenome and Multigene Analyses in the Apicomplexa

As more sequences from more genomes and genes become available, the tendency has been to analyze datasets from more than one genome when carrying out phylogenetic analyses. A number of authors have suggested that a total evidence approach and robust phylogenetic analyses are necessary to resolve some phylogenetic questions in evolution

(Eernisse and Kluge, 1993; Siddall et al., 1997). The use of multiple gene sequences from more than one genome and the advantage of combined data in phylogenetic analyses that confirms or refutes phylogenetic hypotheses have been suggested by some authors (Bull et al., 1993; de Queiroz et al., 1995; DeBry, 2003; Suchard et al., 2003; Gadagkar et al.,

2005). According to Gadagkar et al. (2005), concatenating sequences from different genes appears to be a better method of multigene phylogenetic analysis in contrast to building a consensus tree from analyses involving different genes from different data partitions, irrespective of phylogenetic methods used. Suchard et al. (2003) however suggested the use of a Bayesian hierarchical method to harness the inherent advantages of both concatenated and partitioned datasets to arrive at a more precise evolutionary hypothesis. DeBry (2003) further suggested that the use of likelihood methods combined

38 with model-based analysis is important in dealing with data partitions and combined datasets.

In the phylum Apicomplexa some workers have suggested the usefulness of using multiple genes from different genomes in the study of molecular phylogeny of the phylum (Tenter et al., 2002; Morrison et al., 2004; and Morrison, 2008). The challenge however is that some of the genomes (mitochondrial and plastid) and genes have not been proven to be of universal occurrence in the phylum Apicomplexa (see Morrison, 2008).

Only a few genera of the Apicomplexa have been studied phylogenetically using more than one gene from one or more genomes. Both haemosporid parasites and piroplasms have been studied using all three genomes - nuclear, plastid and mitochondrial (Escalante and Ayala, 1994; 1995; Rathore et al, 2001; Kedzierski et al, 2002; Hagner et al 2007;

Lau et al., 2009; Outlaws and Ricklefs, 2010; Hikosaka et al., 2010; Bhoora et al., 2009).

Martinsen et al. (2008) suggested that previous molecular phylogenetic studies of

Plasmodium based on single genes failed to give unequivocal support to some nodes. A phylogenetic study involving the use of four genes from the three genomes of

Plasmodium and other related genera was better at relating major clades with vector shifts into various Dipteran families and vertebrate hosts (birds, mammals and reptiles) according to Martinsen et al. (2008). Phylogenetic analyses using several genes across the three genomes of Plasmodium of showed that the combined analysis was more robust and supported the monophyly of several malarial parasites (Perkins et al.,

2007). Shirley (2000) suggested that there are four genomes in Eimeria tenella, namely; nuclear, mitochondrial, plastid and double stranded RNA genomes, even though only the first three have been used in molecular phylogenetic studies of the genus Eimeria.

39 Schwarz et al (2009) used nuclear (ITS1, ITS2 and 18S rDNA) and mitochondrial (COI) sequences in assessing composition and polymorphisms in different chicken Eimeria species populations. Plastid large subunit (23S) and nuclear 18S rDNA have also been used to study the possible horizontal transfer of Eimeria species from rodents to bats

(Zhao et al., 2001). Using plastid ORF470 and nuclear 18S rDNA, Zhao and Duszynski

(2001) studied the evolutionary relationships between rodent Eimeria species in order to distinguish two independent extant lineages.

In the family Sarcocystidae, nuclear (28S rDNA, 18S rDNA and ITS-1) and other cytoplasmic genes (Hsp70) have been explored in evaluating the relationships among members of its two subfamilies, Toxoplasmatinae (Hammondia, Toxoplasma and

Neospora and Besnoitia spp.) and Sarcocystinae (Sarcocystis and Frenkelia spp.) (see

Votypka et al, 1998; Mugridge et al., 1999; Slapeta et al., 2001; Slapeta et al., 2003;

Monteiro et al, 2007). Even so, relationships among species in the family Sarcocystidae remain partially unresolved.

It is therefore imperative to use more genes from the three genomes to study the molecular phylogenetics of the coccidia, especially those that pose a threat to man and domestic animals. Molecular phylogenetic studies of members of the Apicomplexa have been based on morphological characters and limited molecular data involving genetic markers, principally from the nuclear genome. Added to these limitations is the problem of taxon sampling in resolving the obvious paraphyly and sometimes polyphyly of members of the Apicomplexa.

40 1.7. The objectives this study was to:

1. Generate new sequences from members of the Apicomplexa with unique sets of

primers that can be subjected to robust molecular phylogenetic methods in order

to investigate the phylogenetic relationships and hypothesis erected for the

phylum Apicomplexa by classical systematics.

2. Investigate utility of individual genetic markers from any of the three genomes

(nuclear, mitochondrial and plastid) in the phylum Apicomplexa as useful

phylogenetic tools.

3. Compare the usefulness of the three genomes in answering current phylogenetic

questions surrounding some important members of the phylum Apicomplexa.

4. Evaluate the usefulness of mitochondrial genes such as cytochrome c oxidase

subunit I gene as a potential DNA barcoding target for species identification

(alpha taxonomy) of members of the Sarcocystidae and Eimeriidae.

41 CHAPTER II: MOLECULAR IDENTIFICATION OF EIMERIA SPECIES INFECTING MARKET-AGE MEAT CHICKENS IN COMMERCIAL FLOCKS IN ONTARIO

(The contents of this chapter have been published as follows: Joseph D Ogedengbe, D. Bruce Hunter and John R. Barta, 2011. Molecular identification of Eimeria species infecting market-age meat chickens in commercial flocks in Ontario Veterinary Parasitology doi:10.1016/i.vetpar 2011.01.0091

2.1. Abstract

A previously described multiplex PCR was evaluated for the identification and prevalence of Eimeria species in market-age commercial chicken flocks in Ontario. The multiplex PCR based on species-specific RAPD-SCAR markers was able to distinguish six available laboratory strains of Eimeria species (E. tenella, E. maxima, E. necatrix, E. mitis, E. acervulina, and E. brunetti) and E. tenella, E. maxima and E. acervulina in unknown field samples, including multiple infections in single reactions. No backyard

(0/77) and 20/360 market-age commercial chickens were oocyst-positive using standard fecal flotation methods. PCR identified Eimeria tenella alone (9/360, 2.5%), E. maxima alone (5/360, 1.38%), E. maxima plus E. tenella (5/360, 1.38%) and E. acervulina alone

(1/360, 0.27%) in market-age commercial broilers. This is the first time the multiplex

PCR has been evaluated in poultry establishments in Canada and may prove a valuable tool in epidemiology on commercial farms.

2.2. Introduction

Coccidiosis is an important of poultry and causes nearly US$3 billion of loss in the industry worldwide (Dalloul and Lillehoj, 2006). Losses include mortality, morbidity and cost of preventative or therapeutic drugs and/or vaccination. In addition, many of the in-feed medications commonly used for prevention of infections with Eimeria species have become less effective because some strains of parasites have developed reduced susceptibility to anticoccidials. New prophylactic anticoccidial medications are not being introduced to market nor are they likely to be introduced in the

42 near future. Collectively, this suggests that coccidiosis is likely to have a greater impact on the profitability of broiler meat production in the future (Williams, 1999).

Identification of different types of Eimeria affecting livestock and poultry is important in instituting control measures to address vaccine efficacy and drug resistance. Various molecular methods of diagnosis have been used in epidemiological investigations of coccidiosis (Morris and Gasser, 2006). Fernandez (2003a, b) developed a RAPD-SCAR

(Random Amplified Polymorphic DNA - Sequence Characterised Amplified Region) based method for the simultaneous identification of seven species of Eimeria infecting domestic chickens. The use of the RAPD-SCAR method of identification of Eimeria species has not, as far as we know, been used in Canada.

The most recent published data regarding the prevalence of coccidia in Ontario was obtained more than 30 years ago and reported on the species of Eimeria causing clinical coccidiosis at that time (Lee and Onderka, 1978). The identification of species of

Eimeria isolated by the authors was based on pathological and morphological details which are variable at best and may be difficult due to overlap of sites within the digestive tract where individual species develop. Our objectives in this limited survey were to obtain representative samples from various poultry farms with a view to assess the multiplex PCR as described by Fernandez et al. (2003b) regarding accuracy of species identification and evaluate the current prevalence of coccidia in chickens in Ontario.

43 2.3. Materials and Methods

2.3.1. Samples

Complete intestinal tracts were collected randomly from chickens slaughtered for meat at a commercial poultry processing facility (360 broilers of average age of 35.5 days

(range 28-54) from large-scale commercial poultry houses) and a custom poultry processing facility (77 older meat birds [over 70 days] from backyard operations) in

South-central Ontario during spring [March - April] and early summer of 2007. Ten intact intestinal tracts (duodenum to vent) per farm (or fewer if submitted lots were less than 10 birds) were collected randomly for a total of 437 samples. Samples were placed on cold packs and transported to the laboratory for further processing.

2.3.2. Sample Processing and Oocyst Preparation

Each intestinal tract was examined macroscopically for lesions consistent with coccidial infections and the contents of the intestinal tract were examined for the presence of coccidial oocysts. First, intestinal contents were flushed from the intestinal tract and suspended in 2.5% potassium dichromate (aqueous) to enhance sporulation of oocysts to a total volume of 50ml. Each emptied intestinal tract was completely opened and examined grossly for the presence of lesions typical of coccidiosis. Areas of suspected coccidial lesions were fixed in 10% buffered formalin and processed normally for histological examination. The intestinal content samples from each intestinal tract were then analyzed for the presence of coccidial oocysts using a standard fecal flotation technique (Reid and Long, 1979). Briefly, 5ml from each sample was pelleted by centrifugation at 1500xg for 5 minutes. The resulting pellet was resuspended in saturated sodium chloride (aqueous), passed through a ~lmm mesh size sieve to remove coarse

44 fecal debris. The resulting filtrate was used in a standard gravity vial fecal flotation using

22x22 mm coverslips. After flotation, the coverslip was mounted on a slide and examined in its entirety for the presence of coccidial oocysts. For oocyst-positive samples, the remaining 45ml of the intestinal tract contents were incubated at 26C for 7 days to permit sporulation to complete. Sporulated oocysts were washed to remove potassium dichromate, concentrated by salt flotation, resuspended in distilled water and then stored at 4°C until DNA isolation.

2.3.3. Multiplex PCR Identification of Coccidia

A multiplex PCR method was used to identify any Eimeria spp. observed in samples. Briefly, oocyst walls were broken by vortexing with glass beads (Ferro

Microbeads; Cataphote Division, Jackson Mississippi, USA) before DNA isolation was accomplished using DNAzol (Invitrogen, Carlsbad CA, USA) according to the manufacturer's protocol. In addition to samples shown to contain oocysts using fecal flotation, 189 additional oocyst-negative samples were processed in parallel for DNA isolation. Isolated DNA (~45ng/sample) from both oocyst-positive and oocyst-negative samples were used as templates in 50ul multiplex PCR solutions containing 1*PCR buffer, 2.5mM MgCb, and one Unit of Taq DNA polymerase (Invitrogen) and seven pairs of species-specific primers for 7 Eimeria species that infect chickens (Fernandez et al, 2003b). The PCR was completed using an MJ mini thermal cycler (Bio-Rad

Laboratories, Hercules CA, USA) according to the PCR parameters described by

Fernandez et al (2003a). Reaction conditions consisted of an initial denaturation at 96° for 5 min followed by 35 cycles of 1 min at 94°and 2 min at 65°, with a final extension step at 72° for 7 min. Amplified products were electrophoresed in submarine 1.5%

45 agarose gels in TAE [Tris-Acetate-EDTA] buffer at 120V for 45 minutes, stained with ethidium bromide and then visualized using UV transillumination.

2.3.4. Statistical Analysis

Mean ages of infected and uninfected birds were determined and considered significantly different using Student t-test if they differed statistically (p<0.05).

Prevalence of Eimeria sp. infections in flocks treated with anticoccidials versus those flocks in which no anticoccidials were used were tested for significance using a chi square test.

2.4. Results

2.4.1. Histological Observations

Histological observations of the cecum, duodenum and jejunum of birds with macroscopically visible lesions demonstrated endogenous coccidial stages typical of

Eimeria species (Fig. 2.1). Typical lesions indicating severe infections with E. tenella were noted in the cecal epithelium of some birds that had bloody, congested ceca.

2.4.2. Prevalence of infections with Eimeria spp.

We found an unexpectedly high prevalence of oocysts in market-age meat chickens from commercial poultry flocks but none from meat birds from backyard flocks

(Table 2.1). Samples from 8 of 36 commercial farms were found to be infected with at least one Eimeria sp. at the time of slaughter and, on these affected farms, at least 25% of birds were shedding coccidial oocysts. Four of the 8 infected flocks had been treated with in-feed anticoccidial medications. There was no significant difference; Student's T-test

(p<0.05; P=4.11) in the mean ages of birds from infected versus uninfected flocks (35.0 versus 35.7 days of age, respectively) nor in the overall prevalence of infections in birds

46 treated (15 farms with 150 birds sampled) with anticoccidial medications versus those that were not (21 farms with 210 birds sampled) 6.0% (n=150); 5.24% (n=210), respectively using Chi Square analysis (p-value = 0.48).

2.4.3. Multiplex PCR Identifications

Laboratory strains of six Eimeria species were successfully identified by distinct diagnostic bands on agarose gels (Fig. 2.2). Comparable PCR products assignable to individual Eimeria species were identified by apparent molecular weight (Fig. 2.3) from all oocyst-positive, intestinal content samples. PCR identified Eimeria tenella alone

(9/360, 2.5%), E. maxima alone (6/360, 1.67%), E. maxima plus E. tenella (5/360,

1.38%) and E. acervulina alone (1/360, 0.27%) in market-age commercial broilers

(Table 2.1). All oocyst-positive samples generated at least one species-specific PCR product using the RAPD-SCAR PCR assay. The few birds showing cecal lesions (see

Fig. 2.1) all had oocysts in their intestinal contents and these were identified as E. tenella using the PCR assay. DNA from none of 189 additional oocyst-negative samples processed in parallel generated any £7men'a-specific PCR products using the same assay.

2.4. Discussion

Published data on the prevalence of Eimeria species in Ontario chickens are limited and dated. A lone report by Lee and Onderka (1978) was limited to diagnostic samples submitted to the Ontario Veterinary Laboratory Services Branch prior to the widespread use of ionophorous anticoccidials. Putative identifications of the causative agents were based on the location and appearance of lesions without definitively identifying the Eimeria species causing the disease. Shirley (1975) discussed the unreliability of using morphological and pathological details in diagnosing oocysts

47 because these features overlap considerably among Eimeria species; this recognition has driven the development of molecular methods for differentiating Eimeria species that infect chickens (e.g. Morris and Gasser, 2006). Recently, molecular methods have been used in an attempt to correlate flock performance with the epidemiology of Eimeria species (Schwarz et al, 2009). The multiplex RAPD-SCAR PCR used in this study is one such method that has not to our knowledge been used on a flock basis to identify chicken coccidia in Canada. The RAPD-SCAR PCR assay was developed by Fernandez et al. (2003a) by designing primers that allow the simultaneous identification of seven

Eimeria species infecting chickens, namely E. tenella, E. maxima, E. acervulina, E. brunetti, E. necatrix, E. mitis and E. praecox (Levine, 1988; Williams, 1999). Two less widely accepted Eimeria species, E. mivati and E. hagani, have been described from chickens but these are not specifically included in the multiplex PCR method used in this study (Reid and Long, 1979; Fernandez et al., 2003a). In addition to the testing of the

RAPD-SCAR PCR assay with laboratory strains of coccidia as described herein, we have successfully used this method to identify different Eimeria sp. isolates from Nigeria and the United (Ogedengbe et al., 2008). This PCR method is relatively cost effective because the diagnosis of all seven species of coccidia is done in a single PCR reaction and is sensitive enough to detect <100 oocysts in a sample (JDO, unpublished observations). Oocysts in fecal or litter samples can be concentrated using standard fecal flotation methods prior to DNA isolation to increase the sensitivity of the method to a few oocysts/gram of fecal or litter material. DNA from infected tissues (when available) would, at least in theory, provide greater sensitivity because of the range of endogenous stages available for sampling; however, the increase in sensitivity would come with a

48 tremendous increase in cost and workload because multiple DNA isolations (or multiple samplings along the gut pooled into a single DNA sample) would be required. Even with multiple samplings, it could be possible to have a coccidial infection present that could be missed unless DNA is extracted from the entire intestinal tract. Finally, 'natural' infections in a poultry house are unlikely to be synchronized and therefore oocysts are likely to be present for the majority of the time a is infected with coccidia. For these reasons, we chose to focus on intestinal contents (and the oocysts that could be concentrated from them) rather than on endogenous stages.

The unexpectedly high prevalence of oocysts in market-age meat chickens is probably attributable to higher stocking density of commercial poultry houses; indoor housing and the likelihood of oocyst buildup in the environment that promotes oocyst contamination of feed or water. In backyard poultry, birds tend to be held at lower stocking densities and with mixed ages. The birds from backyard flocks are likely to be grown for a longer period before being sent for slaughter. Older birds in such environments would likely have acquired immunity to any Eimeria species found in their environment and, as observed in this survey, few, if any, would be shedding oocysts any longer (McDougald and Reid, 1997). Birds sampled from backyard flocks in this study were 10 or more weeks of age, however exact age data were not available.

Prophylactic in-feed anticoccidial usage did not appear to prevent coccidiosis in sampled flocks; both macroscopically visible lesions as well as oocysts were observed in birds shipped from such farms. Reduced susceptibility to anticoccidials has been demonstrated in many Eimeria species on commercial broiler farms and the application of vaccines may be too expensive or impractical in large broiler operations (Williams,

49 1999). Intestinal tract lesions resulting from coccidiosis in market-age birds may make the tissues more fragile and increase the risk of rupture during automated evisceration of

the carcasses; this can increase the risk of fecal contamination of carcasses during poultry processing and may require secondary processing and reinspection that will increase processing costs. More comprehensive molecular epidemiological studies on the prevalence of coccidiosis are warranted to guide control measures and highlight specific

Eimeria species that are found to be most problematic in the commercial broiler industry.

50 TABLE 2.1. DETECTION OF EIMERIA SPECIES IN MARKET-AGE MEAT BIRDS FROM COMMERCIAL BROILER FLOCKS. ALL FECAL FLOTATION-POSITIVE SAMPLES YIELDED SPECIES IDENTIFICATION USING MULTIPLEX PCR. NO MULTIPLEX PCR-SPECIFIC PRODUCTS WERE AMPLIFIED FROM ANY OF THE 189 FECAL, FLOTATION NEGATIVE DNA SAMPLES TESTED. Age of No. of Multiplex PCR Farm chickens Anticoccidials" Fecal +ve b (Days) Samples Eimeria sp. ID N 38 MxB 10 3 Et+Em (2); Em (1) V 38 MxB 10 2 Em (2) Z 28 MxB 10 1 Em(l) H 37 MxB/MoB 10 3 Et(3) W 40 - 10 4 Et(4) E 35 - 10 3 Et (2); Em (1) P 30 - 10 3 Et+Em (3) U 38 - 10 1 Ea(l) F 33 Clin 10 0 - Af 32 MxB 10 0 •- Ag 42 MxB 10 0 - B 34 MxB 10 0 - Aa 34 MxB 10 . 0 - Ab 34 MxB 10 0 - Ac 32 MxB 10 0 - Aj 31 MxB 10 0 - R 45 MxB 10 0 - X 35 MxB 10 0 - D 34 MxB/MonB 10 0 - A 32 - 10 0 - Ad 36 - 10 0 - Ae 37 - 10 0 - Ah 40 - 10 0 - Ai 35 - 10 0 - C 35 - 10 0 - G 32 - 10 0 - I 35 - 10 0 - J 54 - 10 0 - K 28 - 10 0 - L 31 - 10 0 - M 37 - 10 0 - O 45 - 10 0 - Q 37 - 10 0 - s 30 - 10 0 - T 36 - 10 0 - Y 33 - 10 0 - Mean Age 35.53 Total Samples 360 Max Age 54 Min Age 28 "Anticoccidials: MxB - Maxiban® (narasin and nicarbazine); MoB - Monteban® (Narasin); Clin - Clinacox® (Diclazuril) hEimena species. Et - Eimeria tenella; Em - Eimeria maxima; Ea - Eimeria acervulina. The numbers in the flotation and PCR columns are the numbers found positive for both techniques respectively. The numbers in bracket in the PCR result column indicates the number of each identification.

51 Figure 2.1. Histological appearance of the cecal epithelium of a commercial broiler that had visible macroscopic cecal lesions and tested positive for Eimeria tenella using multiplex PCR Panel A - Numerous 2nd generation meronts (arrows) of £ tenella were visible within the disrupted mucosa, Panel B - Other regions of the same cecae contained numerous macrogamonts (ma) and occasional unsporulated oocysts within the inflamed cecal mucosa [Hematoxylin and eosin, scale as marked]

Figure 2.2. RAPD-SCAR-based multiplex PCR products obtained when using single Eimeria species as templates Lanes M, 100bp ladder, 1, Eimeria acervulma (811 bp), 2, Eimeria brunetti (626 bp), 3, Eimeria tenella (539 bp), 4, Eimeria mitis (460 bp), 5, Eimeria maxima (272 bp), 6, Eimeria necatrix (200 bp), C, no parasite DNA control

52 Figure 2.3. Typical agarose gel showing separated multiplex PCR products with positive amplification products for Eimeria tenella (525 bp, upper amplicon) and Eimeria maxima (272 bp, lower amplicon). Lanes: M, 100 bp ladder Marker; 1-9, individual DNA samples from intestinal contents containing oocysts; 10, negative control.

53 CHAPTER III: PHYLOGENETIC POSITION OF THE ADELEORINID COCCIDIA (MYZOZOA, APICOMPLEXA, COCCIDIA, EUCOCCIDIORIDA, ADELEORINA) INFERRED USING 18S RDNA SEQUENCES.

(The contents of this chapter have been submitted for publication as. Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18s rDNA sequences. Journal of Eukaryotic , in preparation)

3.1. Abstract

Phylogenetics studies of the adeleorinid coccidia have mostly been based on morphological details of blood forms of the parasites especially the families

Hemogregranidae and Adeleidae. The position of the adeleorinid coccidia in relation to other members of the Apicomplexa remains an important area of study and the relationships within the group has important implications in host parasite relationships.

We generated new sequences from both monoxenous and heteroxenous adeleorinid coccidia and used robust phylogenetic analyses to study the molecular phylogeny of adeleorinid parasites including their relationships among themselves and within the phylum Apicomplexa generally. All adeleorinid genera were found to form clades in all analyses with the exception that the single Hemolivia sp. was found within a clade containing all Hepatozoon spp. included in the analyses. There was some evidence of host-parasite coevolution between the parasites and their definitive () hosts but much less correlation between the parasites and their intermediate (vertebrate) hosts.

Finer resolution of the relationships among these various adeleorinid parasites will require denser taxon sampling and likely the use of other, faster evolving, genetic loci in concert with 18S rDNA sequences.

54 3.2. Introduction

Members of the phylum Apicomplexa are protists characterized by an apical complex and are exclusively parasitic on hosts that range from marine invertebrates to terrestrial vertebrates. The apicomplexan parasites share ultrastructural features synapomorphic for the phylum, including the conoid, polar ring, apical rings and rhoptries that are collectively called the apical complex (Siddall 1995; Perkins et al.

2000; Adl et al. 2005). They have other ultrastructural features such as a trilaminate pellicle (cortical alveoli), microspores, subpellicular microtubules and micronemes, and frequently a complex multistage life cycle that may include one or more hosts (Adl et al.

2005).

In addition to the recently recognized apicomplexan affinities of the colpodellid protists (Kuvardina et al. 2002), historically recognized 'groups' of apicomplexan parasites include the gregarines, piroplasms, haemosporinids, Cryptosporidia and coccidia. The latter is usually considered to include the tissue coccidia (;

Sarcocystidae), the enteric coccidia (Eimeriorina; Eimeriidae), the adeleorinid coccidia

(Adeleorina; Adeleidae) and the haemogregarines (Adeleorina; various families).

Compared with some apicomplexans parasites, members of the suborder

Adeleorina Leger 1911 remain relatively poorly understood with respect to their biology and taxonomic affinities (Barta 1989; Barta 2000). The monoxenous adeleorinid coccidia infect a wide range of invertebrates (Levine 1988). The vast majority of the heteroxenous haemogregarine parasites have been described only from their gamonts within the blood of their intermediate vertebrate hosts, usually within erythrocytes or leukocytes (Siddall 1995; Smith et al. 2000).

55 The haemogregarines utilize a wide variety of definitive hosts such as leeches,

acarines and insects (Ball and Oda 1971). All adeleorinids are characterized by a typical

complex life-cycle involving at least one (and often numerous) asexual cycles of

merogony followed by gametogony, syngamy and sporogony. Many haemogregarines

appear to have morphological variation in meronts and merozoites that are believed by

many workers to either initiate further rounds of merogonic replication or differentiate

into gamonts (Barta 2000). Gamete formation is via syzygy; association of gamonts prior

to final gamete maturation and fertilization.

Major groups within the Adeleorina include monoxenous parasites belonging to

the genera Adelina, (Adeleidae), Legerella (Legerellidae) or

(Klossiellidae), and heteroxenous parasites in the genera Dactylosoma, Babesiosoma

(Dactylosomatidae); Haemogregarina, Desseria and Cyrilia (Hemogregarinidae);

Hepatozoon (Hepatozoidae); Hemolivia, Karyolosus, (Karyolysidae) (see Barta 2000).

Although some molecular phylogenetic analyses have been attempted to study the

phylogeny of adeleorinid parasites using limited numbers of sequences or taxa;

morphological features, cytopathologic changes to infected host cells and host

associations have been the mainstay of taxonomic classification (Siddall 1995; Smith

1996; Smith et al. 2000; Boulianne et al. 2007; Vilcins et al. 2009). Classifications based

on biology and morphology of the myriad stages of these parasites is fraught with homoplasy-related issues and differences in opinion regarding the weight to give

individual characters. This has led to revisions of various taxa and groupings that has

compounded the problem rather than solved it (Siddall 1995).

56 The choice of 18S rDNA sequences in the present study is a pragmatic one; the gene has been shown to be useful for inferring genus level relationships within the

Apicomplexa and there are more sequences available for comparison with newly obtained sequences than any other molecular target (Fitch et al. 1995; Rubini et al. 2006;

Kopecna et al. 2006). The objectives of this study were to elucidate the phylogenetic position of Adeleorinid coccidia in relation to other parasites within the phylum

Apicomplexa using newly obtained 18S rDNA sequences to test the monophyly of the

Adeleorina and, if monophyletic, evaluate the morphological and life history in the context of this molecular phylogeny.

3.3. Materials and Methods

3.3.1. Parasite Material

Klossia helicina (Schneider 1875) - Oocysts of Klossia helicina (Adeleidae) were obtained by dissecting infected Grove snails (Cepaea nemoralis Linn.) collected at

Lochnager Crater in France (50° 00' 56" N and 2° 41' 50" E). Oocysts were cleaned of debris manually using a De Fonbrune micromanipulator and then suspended in bleach

(5.25% w/v sodium hypochlorite) for 10 minutes to destroy any contaminating host

DNA. After washing in phosphate buffered saline (PBS), oocysts were broken by 3-5 freeze-thaw cycles by transferring the samples repeatedly between liquid nitrogen and a

37C water bath.

Blood parasites of various poikilotherms - Gamonts of Hepatozoon magna

(Grassi & Feletti 1891) Labbe 1899 and Dactylosoma ranarum (Lankester 1882)

Wenyon 1926 were obtained from naturally infected edible frogs, Pelophylax kl. esculentus (Linn.) (syn. Rana esculenta Linn.) collected by hand from the Fium 'Orbo

57 River, Corsica, France (42° 0' 49"N, 9° 24' 21"E). Babesiosoma stableri Schmittner &

McGhee (1961) and Hepatozoon cf catesbianae (Stebbins 1903) Desser, Hong & Martin

(1995) were obtained from Rana septentrionalis Baird 1854 and Rana catesbiana Shaw

1802, respectively, collected by hand from Lake Sasajewun, Algonquin Provincial Park,

Ontario, Canada (45° 35' 17"N, 78° 31' 45" W). Also collected at Lake Sasajewun were blood samples containing gamonts of Haemogregarina balli Paterson and Desser 1976 obtained from the common snapping turtle, Chelydra serpentina serpentina Linn. Finally, two samples of Hepatozoon cf clamatae (Stebbins 1905; Smith 1996) were collected from the blood of green frogs, Rana clamitans (Latreille 1801), collected by hand at

Birge Mills, Ontario, Canada (43° 39' 59"N, 80° 15' 30" W) and the Speed River,

Guelph, Ontario, Canada (43° 32' 51"N, 80° 11' 52" W). For all blood samples, a thin blood film was made from each host animal and stained with Giemsa's stain. Stained blood smears were examined thoroughly using light microscopy to ensure that only a single species of blood parasite was present based on morphometry of intraerythrocytic stages before the remainder of the blood sample was used for DNA extraction.

In addition to parasites that were collected personally, samples containing gamonts of Hemolivia mariae (Smallridge and Paperna 1997) were kindly provided by

Dr. C. Smallridge as dried blood films of experimentally infected skinks, Tiliqua rugosa,

Gray 1825. The parasite was originally obtained from naturally infected T. rugosa collected near Mt. Mary, South Australia (near 33° 55' S; 139° 20' E). Uninfected lizard blood was also made available as a suitable PCR amplification control.

3.3.2. DNA extraction and rDNA PCR Amplification

58 For all samples of hematozoa, air-dried blood smears containing parasites were scraped into lOOul of PBS using sterile scalpel blades. DNA extraction from blood or freeze-thawed oocysts of K. helicina was achieved using a guanidine thiocyanate-based, rapid DNA isolation kit according to the manufacturer's instructions (IsoQuick Nucleic

Acid Extraction Kit, Orca Research Inc., Bothell, Washington).

For amplification of K. helicina DNA, standard PCR reactions with -specific forward and reverse primers (Medlin et al. 1988) were used to amplify near full length rDNA. The resulting PCR amplicons (~1.8kb in length) were gel-purified by separation on a 1.5% agarose gel, purified using a Roche High Pure DNA purification kit (Roche Applied Science, Germany) and then cloned into a pCR2.1 plasmid vector using the TOPO TA Cloning® kit (Invitrogen Corp., Carlsbad, CA). Plasmid isolation was performed using GenElute Plasmid Mini-Prep kit (Sigma, St. Louis, MO). Complete sequences were obtained using Ml3 forward and reverse primers as well as a number of internal sequencing primers including: 366F - AGGGTTCGATTCCGGAG,

555F - GTGCCAGCRGCCGCGG, 571R - ACCGCGGCKGCTGGC,

1125F - GAAACTTAAAKGAATTG and 1139R - ATTCCTTTRAGTTC (see Elwood et al. 1985) or NS2 - GGCTGCTGGCACCAGACTTGC and

NS3 - GCAAGTCTGGTGCCAGCAGCC (see White et al. 1990; equivalent to 4558 of

Mathew et al. 2000). PCR-based, Sanger sequencing reactions and electrophoresis of the resulting products were performed using ABI Prism 3730 and 3100 DNA sequencers by the Molecular Biology Section (Laboratory Services Division, University of Guelph). All clones were sequenced completely in both directions and chromatogram-based contigs

59 were generated using Geneious 5.1 and later versions (Drummond et al. 2010) to provide high quality sequences for further analyses.

Amplification of the nuclear 18S rDNA of the parasites in blood samples from various poikilotherms was not possible using universal eukaryotic primers because of the large surplus of contaminating host DNA that would be amplified preferentially. Instead, two sets of primers, each combining a eukaryote-specific primer with an apicomplexan- specific primer were used to generate partial, but substantively overlapping, rDNA fragments. The 'A fragment', consisting of the first ~1.35kb of the nuclear 18S rDNA, was obtained using a 'universal' forward primer (Medlin et al 1988) with a parasite- specific reverse primer 18AP1488.R 5'-CGGAATTAACCAGACAAATC-3', (Wozniak et al. 1994). The 'B fragment', consisting of the last ~1.65kb of the nuclear 18S rDNA, was amplified using a parasite-specific forward primer (NS3 of White et al. 1990, see above) with a 'universal' reverse primer (Medlin et al. 1988). The resulting PCR products were separated using agarose electrophoresis, purified, cloned and sequenced as described above for K. helicina. Complete rDNA sequences of the blood parasites were obtained by combining the largely overlapping cloned A and B sequences to provide a contiguous, complete rDNA sequence. In most cases, fragments A and B provided unambiguous complete nuclear 18S rDNA sequences (i.e. 100% identity over the overlap region of approximately 1000 bases) but, inevitably, clone to clone sequence variation was present in some parasite rDNA sequences. In these cases, unique sequences were treated as paralogs and analysed separately (see below). All sequences have been submitted to GenBank.

3.3.3. Phylogenetic analyses

60 In addition to the new sequences obtained in the present study, sequences from a

large number of parasitic apicomplexan taxa were included in the phylogenetic analyses

(Table 3.1). New and existing sequences were aligned using Clustal-W (Larkin et al.

2007) implemented in the Geneious (Ver. 5.1) bioinformatics software package. This primary alignment was then optimized by eye using a staggered alignment method

(Barta, 1997; Chenna et al. 2003). For initial testing of monophyly of the adeleorinid taxa among other apicomplexan parasites, all sequences in Table 3.1 were used to perform the phylogenetic analyses. After this global analysis, 6 sequences from monoxenous parasites in the family Adeleidae were used as a functional outgroup to root 72 sequences from heteroxenous adeleorinid taxa in the families Hemogregarinidae, Karyolysidae,

Dactylosomatidae and Hepatozoidea for a total of 78 sequences representing about 21 named or unnamed haemogregarine taxa.

PAUP Ver 4.10b (Swofford, 2003) was used to analyze the dataset using both maximum likelihood (ML) and maximum parsimony (MP) criteria. Bayesian analysis was performed using MrBayes Ver. 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and

Huelsenbeck 2003) on the same dataset. Nucleotide substitution models used as a priori assumptions for the ML and Bayesian analyses were generated using MrModeltest 2.3

(Nylander 2004). The model and parameters generated using Modeltest for each dataset were used for all ML and Bayesian analyses performed on each dataset. All Bayesian analyses used the following parameters: number of generations=106; number of runs=2; sample frequency=100; burnin value=105; number of chains=4; and temp=0.20.

For all MP analyses, characters were considered unordered and changes were given equal weighting. During all MP analyses, bootstrap analyses (100 replicates) were performed

61 using the heuristic search algorithm within PAUP. Strict consensus trees generated from all analyses were visualized using Treeview (Page 1996).

3.4. Results

3.4.1. Parasite Sequences:

Ten complete or partial 18S rDNA sequences from adeleorinid parasites were obtained as follows (length in bp; GenBank accession number): Haemogregarina balli

[1817 bp; HQ224959], Hemolivia mariae [1649 bp; HQ224961], Dactylosoma ranarum Clone 1A22 [1810 bp; HQ224957], Dactylosoma ranarum Clone 1B16

[1810 bp; HQ224958], Hepatozoon magna [1817 bp; HQ224960], Hepatozoon cf catesbianae [1810 bp; HQ224954], Hepatozoon cf clamatae Clone B_l [1655 bp;

HQ224962], Hepatozoon cf clamatae Clone B_2 [1655 bp; HQ224963], Klossia helicina

Clone 2_6 [1799 bp; HQ224955] and Klossia helicina Clone 4_3 [1799 bp; HQ224956].

3.4.2. Phylogenetic Analyses:

Initial analyses included a wide range of taxa to establish the monophyly of the adeleorinid taxa among the 85 apicomplexan eukaryotes included in the analysis (see

Table 3.1). For this dataset containing near full-length 18S rDNA sequences (~1800bp) from a wide range of apicomplexan taxa, likelihood settings from best-fit model were

(GTR+I+G) selected by hLRT in MrModeltest 2.3 (Nylander, 2004). The alignment totaled 3906 bp in length (including spaces to permit staggered alignment - see Barta,

1997). All characters had equal weight and 1941 characters were constant. 817 characters were parsimony uninformative. Number of parsimony informative characters was 1148.

Both ML and MP gave similar tree topologies. The MP consensus tree length was 4692 with a consistency index (CI) of 0.61, CI excluding uninformative characters of 0.51 and

62 Retention index of 0.81 (tree not shown). Bayesian analysis was performed using the following parameters; Lset; nst=6; rates=gamma. In all analyses, monophyly of the 25 adeleorinid taxa included in the analysis was strongly supported (see Fig. 3.1).

For more detailed analysis of the adeleorinid ingroup, 78 haemogregarine sequences were rooted by 6 sequences from monoxenous adeleorinid parasites

(Adeleidae - species of Adelina and Klossia) that acted as a functional outgroup.

Mrmodeltest2.3 suggested the general time reversible with gamma rate of distribution model as best for the data, Lset Base=(0.3054 0.1697 0.2217) Nst=6 Rmat=(1.1072

4.6577 1.6225 0.76014.0870) Rates=gamma Shape=0.7973 Pinvar=0.3680. All three phylogenetic analyses methods (ML, MP and Bayesian analyses) were used and provided near-identical tree topologies. MrBayes settings for the best-fit model (GTR+I+G) were selected by hLRT in MrModeltest 2.3 with the following parameters: Lset nst=6; rates=invgamma. The tree generated using Bayesian analysis is shown (Fig. 3.2). For

MP analysis, 1903 characters of equal weights were in the dataset. Of these, 1336 characters were invariant, 199 characters were variable but phylogenetically uninformative, and 368 characters were parsimony informative. The MP tree length was

1014 with a consistency index of 0.70 (tree not shown).

The haemogregarine taxa included in the ingroup analysis together formed a monophyletic group with strong branch support for a trichotomy that consisted of: i)

Haemogregarina balli; ii) 3 sequences from the family Dactylosomatidae; and iii) a large clade consisting of all Hepatozoon sp. sequences (Hepatozoidae) as well as a single sequence from Hemolivia mariae (Karyolysidae). However, the genus Hepatozoon was found to be paraphyletic if the genus Hemolivia is considered valid. The sole

63 representative of the genus Hemolivia for which sequence data were available, Hemolivia mariae, was found within a large clade of Hepatozoon species that use ticks as their definitive hosts (where known).

Within the strongly supported Hepatozoon/Hemolivia clade described above, there was a large, early branching clade with strong support (1.00) that included a number of

Hepatozoon species infecting carnivores that use ixodid ticks as their definitive host (H canis, H. americanum, H. ursi, H felis and an unnamed Hepatozoon sp. from a pine marten [EF222257]). A Hepatozoon sp. from a water python (EU430234) formed a sister taxon to Hemolivia mariae with 0.63 support; this monophyletic clade was supported in all analyses. Parasites from marsupials from Australia and the Americas formed a monophyletic grouping with 0.99 support; the suspected, but not demonstrated, definitive hosts for these Hepatozoon spp. are all ixodid ticks. A large clade of Hepatozoon spp., with 0.96 support, included parasites transmitted by a wide range of invertebrate definitive hosts; the vertebrate hosts infected by these parasites ranged from amphibia to mammals (Fig. 3.2, Table 3.1).

64 mColpodella tetrahymenae AF330214 t Voromortas pontica AF280076 m pontica AYQ78092 „Cbromeravelia DQ174731 •""'""^""" T RM11DQ174732 Colpodella edax AY234843 Dtnoftagellattts ,Lessardia elongata AF5211GQ Prorocentrum minimum AY421791 Scrippsiella nutncuh U52357 Akashiwo sangumea EF492486 m- »?v at iniy~ir&4if r-A if * AYiMSfc?

iV< i is< aju f is?s < ^¥^54 ^ 0 83, 11 ijiiU * r. 4Wifi P U 1 A J1 ll i? c? 0< sff DO^i ss 4 /H^r ciWi O/* Cryptosporidium wram AF115378 , Cryptosporidium meleagndis AF112574 Af 112571 Cryptosporidium parvum AF 108865 Cryptosporids • Cryptosporidium sp K1AF108860 Cryptosporidium parvum AF108864 _^^B Cryptosporidium fells AF 108862 LM, Cryptosporidium sp EGKAF513227 IMWMWMM Hepatocystis sp £U400394 • P/asmori/um falciparum M19172 Hemosporinids Babesia rodhami DQ641423 8afoes(a/Wia heist no tSMXZWfb j~ Adelines bambarooniae ^4940^8

I & Acei mi an barm, < e Af 494059 &t?h*.i stabler/ NQ2249B1 .otf? D aylostoma ranarum HQ2249S7 Ji o

%=ss» fiormotjregarmcf both HQ2249S9 \, g lippatoiaon felii AYW8681 Hepatoioon felt* AYS20232 "Ikpatoiocn tunpura iMAbl&t? mmw%

( ™H^paf2Gon ctyogbor EriS?822 ileptttaioon -;p A8J81S04 Hffs«f02-Ci??J sp AYb0062S Nepafo/Gon «-p AYb006«>6 iHepatoioon magna HQi>M3foO Hipatazoon cattihianae AF130361 tiepatoroon rtemitarts HQ?2496-SR( U JtepatMoan damttam HQ2219b?CL>

:H*>pato?oon caWsbwrtae I1Q.224954 mGoussia janae AY043206 2

w(5ouss/o neglecta FJ009242

MGOUSSJO sp Su/oFJ009243

B noellert FJ009241

mGoussia metchnikovl FJ009244 zaChoteoeimeria sp AY043207 1 ^Isospora robini AF080612 aEmteria faiaformis AF080614 Etm«rttd& hEwnena nieschulzi U40263 mCydospora sp U40261 &Eimeria acervutina U67115

mCimeria tenella U67121 m Toxoplasma gondii EF4/2967 %Neotpora sp HP U3 /34S %Neospora camnttm AI271354

wCystoiso%pora belli DQ060674

mCystoisospora sins U9/523

MCystoiso-,pora ahioens AF0293Q3 pSarcocystis mucosa AF10967S

m lorcotystfs iacartae AY015113 ^frenkeiio ghreoli AF009245 Sarcocystit &,( renketta microti AF009244

m$arcocy$tis scandtnavtca EU282032 teSarcocystis rang: E:F05fc011 s&Sarcocystis grucneri EF0S6010 saSarcocystis sp HM050622 AB2S7157 '&,Sarcacystts rartaifen EF056016

Figure 3.1. Bayesian phylogenetic analysis using 85 18S rDNA sequences from members of the Apicomplexa including the adeleorinid coccidia.

65 r—Klossia helictnaA'5 DH Cepaea rxmorahs (Gastropoda - ) *— Klossla hellctna 26 DH Cepaea nemoralis (Gastropoda - grove snail) AdelmadtmidiataDQQ96Z2$ DH Scolopendra cmgulata (Crulopoda- centipede) Adeltnabambaroonlae AF494058DH Dermoleplda albohirtim (Coleoptera greyback canegrub) Adelina gryllt DQ096S36DH Gryllus btmaculatus (cncket) Adelma bambaroomae AF4Q4Q59DH Dermoleplda afbotortum (Coleoptera - greyback canegrub) Haemogregarina baill DH Placobdella (leech) IH CTKJ/dra'scr^enI/niHRepUlia-snappinglurtte ) BafcejrosomaJWfctery DH Da sjeroWetor (leech). IH Jfcmspp (Anphibia-green, bull or mink frogs) • Dactplosomaranarum IA22DH Desserobdella Qeecb) iH ft»wesciitenta (Amphibia - European water ) Dactylosoma ranarum IB 16 DH DesseroWeita (leech), [H FanaesculerSa (Amphibia - European waler frog) \QSffitpctSx0cmf2hs AY62Q232DH Fhiptcephalus sp , IH .PC/M rotus (Mammalia - ) itfepatozoonjelis AY62Z6BIDH Fhtpicephahx sp . IH Ms eofus (Mammalia -cats)

]7)i - HepatozoonapEF222257DH ?(ftodcs?XIH Mirtes wwrKs(Mammalia -pinemarten) • Hepatozoon ur si EU041717DH ffaemaphysalis spp, IH Ursui WM^aJsja;wnfcus(Marnmalia-bear) : ife^ofcccowure/EU0'I1718DH Hasmophysalis spp ,IH tfaustAjbeftmausjtopowcijj(MamnBlia-bear) —ffepatozoon amenctmumAF176836DH Amblyommamaculatum, IH Cartspmiliarts (Mammaba -) fliyittfozoojispAY461377 DH 1 (Amblyommaaureolatuml) IH Dusicyon thous (Mammalia -ciab-eaung fox) »•» Hepatozoon cflwsDQlU754DH Fhiptcephalus sanguineus, IH Caws Jamtltaris (Mammalia - dog) 77epirfceocincaiHHEU289222DH Fhiptcephalussanguineus, IH CattiJjfiwi/JiflW (Mammalia-dog) — Hepatozoon cants AF176835DH Rhtpicephalus sanguineus, EH Caws ,#wiri;Gm (Mammalia-dog) ffe/)trtazootteGS vuh?c; (Mammalia - fox) ffepatozoon cants AY461375 DH ? (Amblyommaaureolatum?),^. Dusicyon thous (Mammalia - crab-eating fo* .OCf ffepatozoon cams DQ43QS41 DH Fhiptcephalus sanguineus, EH Ku/pes vuJ^ei (Mammalia - fox) * ffepatozoon cants AY461376DH ? (Amblyomma aureolatum?), !H Aeuddqpexgyttwocercrus (Mammalia- fle^(rfozoo«coMfsGU371446 DH Fhiptcephalus sanguineus, IH fuJ/«i vu/^es (Mammalia - fox) — ffepatozoon cams GU371451 DH Fhiptcephalus sanguineus, IH J'u/pes vwJpcs (Mammalia - fox) - Hepatozoon cants GU37I450DH Wi^ce^Mivs scmgu/weuj. IH Vuipes vuipes (Mammalia - fox) 'Hepatozoon cants GU3714S2DH Rhtpicephalus sanguineus, IH ^u/pej wi/pas (Mammalia - fox) ffepatozoon cams GU371448DH Fhiptcephalus sanguineus, !H JWpes vuipes (Mammalia - fox) Hepatozoon cams GU371447DH Rhtpicephalus sanguineus. IH fWpes vuipes (Mammalia - fox) • Hepatozoon cants GU371449DH Fhiptcephalus sanguineus, IH Vuipes vuipes (Mammalia- fox) Hepatozoon cants GU376457DH Fhiptcephalus sanguineus, IH fuf/«s VU/JJSJ (Mammalia - fox) Hepatozoon cants GU376454DH Fhiptcephalus sanguineus. IH HJJ^CS WJJ^CS (Mammalia - fox) Hepatozoon cants GU376456DH Rhtplcephahis sanguineus, TH Vuipes vuipes (Mammalia - fox) "ra. Hepatozoon canis GU376453DH Fhiptcephalus sanguineus, IH Vuipes vulpzs (Mammalia - fox) Hepatozoon canls GU376455DH Fhiptcephalus sanguineus, IH Vuipes vuipes (Mammalia - fox) • Hepatozoon cants GU37G458DH Fhiptcephalus sanguineus, IH Vuipes vuipes (Mammalia - fox) • ffepatozoon sp EU430236 DH 7(Ani'iTOmwii3/mhr/srtuni'?), IH L;as/sjijsnji(Replilia brown water python) - ffemolivta mariae Perkins DH Amblyomma limbatwn, IH riltqua rugosa (Rtphka - Australian sleepy lizard) oo I- ffe/>£jtazoo«spDG2FJ71!?814DH ? (Ixodes neuquenen!.ls?),\U Dromtaqpsgjiroldes(Mammalia Momto del Monte[marsupial]) ffepatozoon spDGl FJ71S813DH ?(Ixodes neuquenensltf), IH Dromlclops gllroldes (Mammalia -Moruto dd Monte [marsupial]) Hepatozoon sp ZW30237DH ? (Ixodes?), IH. Sarcophtlus tetrrtslt(Mammali a - TastmrnanDewl) R ffepatozoon spEU430238DH 7 (Ixodes?), EH SareopWus Wr/s/j (Mammalia- Tasmaman Devil) Hepatozoon cfperarmlis EF152226DH ? (kodesf), IH Isoodonobesulus (Mammalia-SouthernBrown Bandicoot) •" Hepatozoon cfperamelts EF152227 DH ? (Ixodes7), EH Isoodon obesulus (Mammalia - Southern Brown Bandicoot) - Hepatozoon cfperamellsZFl52230DH 7 (Ixodes?), IH Boodon obesulus (Mammalia -SouthemBrownBandieoDt) —Hepatozoon cfperamelisZFl 52228 DH 7(/;codes7), IH Isoodon obesulus (Mammalia -Southern Brown Bandicoot) •Hepatozoon efperameJJs EF152229DH ? (Ixodes?), IH Isoodon obesulus (Mammalia - Southern Brown Bandicoot) Hepatozoon cf peromelia EF152220DH ? (Ixodes^), IH Isoodon obesulus (Mammalia - Southern BrownBandicoot) — ffepatozoon cfperamellsEV 152225 DH ? (Ixodes?), IH /icodon ofccsuJus (Mammalia - Southern Brown Bandicoot) • Hepatozoon cfperamelts EF152219DH ? (Ixodes?), IH ZsixvfcnotesiJiJi^arnmalia-SouthernBrownBandicoot) Hepatozoon cf peramelts EF15222I DH ? (Ixodes?). IH Isoodon obesulus (Mammalia- Southern BrownBandicoot) Hepatozoon cf peramelts EF152222DH ?(ftodes?),IH Isoodon obesulus (Mammalia- Southern Brown Bandicoot) ffepatozoon cfperamelts EF152224 DH 7 (Ixodes?), IH Isoodon obesulus (Mammalia-Southern Brown Bandicoot) ffepatozoon cf perarmlis EFE52223DH ?(hodes?),\H Isoodon obesulus (Mammalia - Southern Brown Bandicoot) He^i3fceoo>ispEU43Q234DH Amblyommaflmbrlatum, IH Va/anuspanoptes (freptilia -Argus monitor) •ii in i •' Hepatozoon sp AF297085 DH Ambtyomma/Aponomma sp (ticks), IH Botga irregularis (Reptiha - brawn tree ) • flc^orozoontn'orgborEF157822DH mosquito (exp tl), IH Jettonreg/iJj(Reptilia-python) ^^™-FT^WIOZOOJI sp AB 181504 DH ? (/?), IH Bandicota mdica (Mammalia -Greater Bandicoot rat) HF • ffepatozoons p EF222259DH 1 (Megabothra sp)? ,IH Sclurus vulgaris (Mammalia - squinrel) - ffepatozoon cferhardovae?>V\ AY600626DH ? (Megabothrts turbldus?), IH Oethrlonomys gareolus (Mammalia -bank vole) * Hepatozoon tferhwdovai BV2 AY600625DH 7(A&gntoJftm ajrifdUi ?), IH C7ertTO«o»TtfS gtoeoiuj (Mammalia-bank vole) • J7epofcizooMspFJ7198l7DH 7,IH XiTOrt^T oJ/iwceus (Mammalia -olive grass mouse)

wHepatozoon spFJ719818 DH 7flH Abrothrtx ohvaceus (Mammalia - olive grass mouse) pWly>aIC(!ooj]spFJ7198i9DH ?,IH Abrothrix oltvaceus (Mammalia - olive grass mouse) jHepatozoon sp FJ7198E6DH? , IH Abrothrix ohvaceus (Mammalia - olive grass mouse) * ffepatozoon $p?lll9$\5V>K ?.IH Abrothrtx olivaceus (Mamiraiia - olive grass mouse) • Hepatozoon magna DH culiane mosquitoes (presumed), IH Jtoweicutoita (Amphibia- Frogs) m Hepatozoon cfclamatae DH culiane mosquitoes, IH Jfanactoii/tam (Amphibia- Frogs) » ffepatozoon catesbianae AF130361 DH culiane mosquitoes; IH Fma spp (Amphibia -Frogs) V ffepatozoon cf catesblanae DH culiane mosquitoes, IH Fcma cotesi?etana(Anipta,oia- Frogs) r Hepatozoon catesbianae AF176837 DH culiane mosquitoes. IH J4maci3teji>eKmtf(Amphibia-Frogs) ®^Hepatozoon zfcatesblanae DH culianemosqutoes, IH ROTOcatesbetana(Amphibia-Frogs)

Figure 3.2. Ingroup Bayesian phylogenetic analysis using 78 18S rDNA sequences from members of the adeleorinid coccidia showing posterior probabilities for the branching order as well as each parasite's definitive host(s) where known.

66 3.5. Discussion

Classical systematics of the Apicomplexa has been problematic because of the

variability to which morphological details are subject and the lack of extensive molecular

phylogenetic studies of individual groups within the phylum. In the present study, using

18S rDNA sequences and a variety of phylogenetic reconstruction methods, we were able

to demonstrate that the monoxenous adeleorinid coccidia (Adeleidae) and heteroxenous

haemogregarines (Hepatozoidae, Dactylosomatidae, Karyolysidae and

Haemogregarinidae) form a well supported, monophyletic clade of apicomplexan

parasites. All of the parasites within the suborder Adeleorina are united biologically by

demonstrating syzygy (association of gamonts prior to gamete maturation and syngamy)

during their life cycle (Barta, 2000). The gregarines (Gregarinia) and piroplasms

(Piroplasmida such as Babesia and Theileria species) likewise share this biological

feature suggesting that syzygy may be an ancestral feature of apicomplexan protists.

Biologically, syzygy may be a means of ensuring availability and proximity of mature

male and female gametes during infections that are not otherwise synchronized (e.g.

gregarine development in the digestive tracts of invertebrates).

In the analysis including a wide range of apicomplexan taxa, the adeleorinid

coccidia (both monoxenous and heteroxenous) were seen to form a well supported clade that formed a trichotomy with parasites in the Aconoidasida (haemosporinids and piroplasms) and the eimeriorinid coccidia. The gregarines and Cryptosporidia formed a monophyletic group that formed the sister group to these remaining apicomplexan taxa.

The 100% support for the sister group relationship between the gregarines and

Cryptosporidia was higher than in some previous analyses that found no support for the

67 Cryptosporidium and gregarine node (Perkins and Keller, 2001) or lower support for this node (e.g. 64% support of Carreno et al., 1999). Having confirmed monophyly of the adeleorinid coccidia among a range of apicomplexan taxa, the monoxenous adeleorinid sequences were used as a functional outgroup for a more detailed analysis of the heteroxenous adeleorinid blood parasites.

The ingroup analysis, that included all available sequences from adeleorinid parasites greater than 799bp in length, demonstrated that the current taxonomic groups found within the blood-dwelling adeleorinid parasites (haemogregarines and dactylosomatids) were reasonably well supported. The haemogregarines include at least

400 described species belonging to four principal genera (Jakes et al. 2003):

Haemogregarina Danilewsky, 1885; Labbe, 1894; Hepatozoon Miller, 1908; and Cyrilia Lainson, 1981. A fifth haemogregarine genus, Hemolivia, was described by

Petit et al. (1990) to acknowledge the unique stellate oocyst formed by the type species

Hemolivia stellata, and the sixth genus, Desseria, was erected by Siddall (1995) to accommodate piscine haemogregarines that lacked intraerythrocytic merogony. Until recently, many haemogregarine species (members of the Haemogregarinidae,

Karyolysidae or Hepatozoidae) have been misclassified taxonomically because of a

(necessary) reliance on morphology of gamonts in the blood of their vertebrate intermediate hosts and their associations with these hosts as principal taxonomic criteria

(Siddall 1995; Zhu et al. 2009). In all cases, assignment of any 'haemogregarine' parasite to the correct genus ultimately requires knowledge of the nature of the sexual reproduction of the parasite in its definitive host (a blood-feeding invertebrate where known). The present study suggests that 18S rDNA sequences may be useful adjuncts

68 for identifying generic affiliation of a particular parasite without prior knowledge of the sporogonic development of these parasites. Certainly Hemolivia and Hepatozoon spp., united by transmission that is via the ingestion of the definitive host, together formed a monophyletic grouping in the ingroup analysis indicating that 18S rDNA sequences also support this as a natural taxonomic grouping. To our knowledge, the sporogonic development of the Hepatozoon sp. from the water python that formed a monophyletic clade with Hemolivia mariae has not been observed. This suggests that if the sister relationship of this Hepatozoon sp. is accurate; this parasite of water pythons may actually belong to the genus Hemolivia rendering the genus Hepatozoon paraphyletic.

Hemolivia mariae is transmitted by ticks in the genus Amblyomma and ticks of the same genus are likely definitive hosts for the 'Hepatozoon' sp. of the water python in northern

Australia. The formation of relatively large polysporocystic oocysts by Hepatozoon,

Klossia and Adelina species appears to be a symplesiomorphic character. The haemogregarines that undergo sporogonic development within cells lining the digestive tract of invertebrates (without the formation of a resistant oocyst structure) such as

Dactylosoma, Babesiosoma and Haemogregarina species were shown to form a monophyletic group in the present analysis although insufficient taxon sampling precluded supporting the monophyly of their respective familial and generic taxonomic affiliations.

The ingroup analysis suggests a possible evolutionary history for the group; the ancestral form of the adeleorinid parasites was likely similar to the monoxenous Adelina and Klossia spp. with the formation of large, resistant, polysporocystic oocysts. These parasites likely infected initially the digestive tract and later exploited deeper tissues in

69 their definitive hosts. The heteroxenous Hepatozoon spp. retained these polysporocystic, resistant oocysts and depended on the ingestion of their hosts for transmission. Hemolivia and Karyolysus spp. (not sampled in the present work) further exploited the tissues of their definitive hosts but still depend on ingestion of this host. Karyolysus species (not sampled in the present work) could be considered an intermediate stage in this evolutionary process because Karyolysus spp. do not form resistant oocysts like

Hepatozoon species and have sporozoites that migrate into the tissues of their definitive host but Karyolysus spp. are still transmitted by ingestion of their definitive host (they have not invaded the salivary glands to permit transmission during blood feeding).

Ultimately, other haemogregarines evolved to be transmitted via the bite of their definitive hosts and vectors, such resistant oocysts became unnecessary (e.g. parasites in the Haemogregarinidae and Dactylosomatidae). In this latter monophyletic clade of parasites (Fig. 3.2), the sporozoites move out into the tissues of the definitive host and ultimately infect the salivary glands where sporozoites (in the case of Haemogregarina and Cyrilia spp.) or merozoites (Babesiosoma and Dactylosoma spp.) can be transmitted to the next susceptible vertebrate host. Additional taxon sampling may provide support for this proposed evolutionary scenario.

Even within a single genus, Hepatozoon, there was host-parasite association of the various Hepatozoon species with their definitive hosts. Within the large

Hepatozoon/Hemolivia clade, species that used ticks as definitive hosts branched early compared to Hepatozoon species that utilize other invertebrate definitive hosts such as , or mosquitoes. Strict definitive host-parasite coevolution was not observed in this group but this lack of resolution may be addressed through the greater taxon

70 sampling of these parasites and, perhaps, a more quickly evolving alternative gene target

such as cytochrome c oxidase subunit I (COI). This mitochondrial protein-coding gene

was recently shown to be equal to and perhaps better than 18S rDNA sequences for

defining clades among closely related coccidian parasites (Chapter 4; Ogedengbe et al.,

2011). The results of the present study reinforce the idea suggested by Smith (2000) and

Siddall (1995) that the genus Hepatozoon is almost certainly paraphyletic and that these parasites may be more accurately assigned to a number of distinct genera that are more uniform biologically.

In summary, we used nuclear 18S rDNA sequences in our analyses and have

confirmed the monophyly of apicomplexan parasites in the suborder Adeleorina. The monoxenous adeleorinid parasites (members of the Adeleidae) populated the early branches within the suborder. The heteroxenous parasites formed a monophyletic

grouping and the existing haemogregarine families each formed monophyletic groups

(although the relatively branching order among the various families was not resolved) if the single Hemolivia sp. sequence was not considered. The placement of Hemolivia mariae (Karyolysidae) within a large clade of Hepatozoon spp. suggests that this latter genus may need to be split into several new genera that better reflect the biological diversity currently residing within the genus Hepatozoon. Alternately, the erection of the genus Hemolivia may need to be re-examined to decide if the generic characters used to define this taxon are justified; perhaps the biological variation of the existing genus

Hepatozoon is broad enough to encompass species currently placed in the recently described genus Hemolivia. Definitive placement of the adeleorinid coccidia within the phylum Apicomplexa and the relationships among the various families within the

71 suborder Adeleorina will invariably require additional gene sequences, probably including sequences data from the mitochondrial or plastid genomes of these parasites, and a more varied selection of parasite taxa, especially within the families

Haemogregarinidae, Dactylosomatidae and Karyolysidae.

72 TABLE 3.1. ORGANISMS ANALYZED PHYLOGENETICALLY USING NUCLEAR 18S RDNA SEQUENCES. GENBANK ACCESSION NUMBERS, CURRENT TAXONOMIC POSITION AND HOST(S), IF ANY, ARE PROVIDED. Species GenBank Taxonomic Definitive Host" Intermediate Accession Position8 Hostc Prorocentrum AY421791 ; n/a n/a minimum ; Prorocentraceae Lessardia elongata AF521100 Dinophyceae; n/a n/a ; Podolampaceae Akashiwo sanguinea EF492486 Dinophyceae; n/a n/a ; Gyrnnodiniaceae Scrippsiella SNU52357 Dinophyceae; n/a n/a nutricula Perinidales; Scrippsiella. Chromera velia DQ174731 n/a n/a Chromed da sp. DQ174732 n/a n/a PvMll Colpodella AF330214 Apicomplexa; n/a n/a tetrahymenae Colpodellida Colpodella edax AY234843 n/a n/a pontica AF280076 Apicomplexa; Voromonadida n/a n/a Voromonas pontica AY078092 Hepatocystis sp. EU400394 Apicomplexa; Culicine Primates: Aconoidasida; mosquitoes: Cercopithecus sp., Haemospororida Papio sp., fulvithorax, C. Hylobates sp. adersi Plasmodium M19172 Anopheline Humans falciparum mosquitoes Babesia equi Z15105 Apicomplexa; Ticks: Equines Z15104 Aconoidasida; Dermacentor sp., Equines Piroplasmorida Rhipicecphalus sp., Hyalomma sp. Babesia rodhaini DQ641423 Ticks (Mus musculus) Babesiafelis AF244912 Felines Babesia leo AF244911 Theileria sp. L19081 Ruminants Theileria annulata DQ287944 Ticks: Hyalomma Cattle, water sp. buffalo Hepatozoon AY461377 Apicomplexa; Ticks Mammals curupira Conoidasida; Hepatozoon canis DQ439543 Coccidiasina; Ticks: Canines

73 Venezuela 1 Eucoccidiorida; Rhipicephalus Adeleorina; sanguineus Hepatozoon canis DQ439540 Hepatozoidae Ticks: Canines Venezuela 2 Hepatozoon felis AY628681 Ticks: Felines Rhipicephalus sanguineus Hepatozoon felis AY620232 Ticks: Felines Rhipicephalus sanguineus, Ixodes tasmanil Hepatozoon sp. AF297085 Unknown Brown tree snake Boiga {Boiga irregularis) Hepatozoon EF157822 Mosquitoes: Culex Royal python ayorgbor quinquefasciatus {Python regius) (experimental) Hepatozoon sp. AB181504 Ticks: Bandicoot rats HepBiCMOOl Haemaphysalis (Bandicota indicd) bandicotal Hepatozoon sp. BV2 AY600625 Ixodes ricinusl Bank vole Hepatozoon sp. BVl AY600626 {Clethrionomys glareolus) Hepatozoon magna HQ224960 Unknown, Edible frog presumed to be a (Pelophylax kl. culicine mosquito esculentus) Hepatozoon AF130361 Culex territans and Bullfrog (Rana catesbianae other Culex spp. catesbeiana) Hepatozoon cf HQ224954 catesbianae Hepatozoon cf HQ224962 Green frog (Rana clamatae clone B1 clamitans) Hepatozoon cf HQ224963 clamatae clone B2 Haemogregarina HQ224959 Apicomplexa; Glossiphoniid Common snapping balli Conoidasida; leeches: turtle (Chelydra Coccidiasina; Placobdella serpentina) Eucoccidiorida; parasitica, Adeleorina; Placobdella ornata Haemogregannidae Hemolivia mariae HQ224961 Apicomplexa; Ticks: Amblyomma Tiliqua rugosa Conoidasida; limbatum (skink) Coccidiasina; Eucoccidiorida; Adeleorina; Karyolysidae

74 Dactylosoma HQ224957 Apicomplexa; Glossiphoniid Edible frog ranarum 1A22 Conoidasida; leeches: (Pelophylax kl. Dactylosoma HQ224958 Coccidiasina; Desserobdella esculentus) ranarum IB 16 Eucoccidiorida; picta Adeleorina; (experimental) Dactylosomatidae Adelina AF494058 Apicomplexa; Greyback cane n/a bambarooniae Conoidasida; grub {Dermolepida Ba3:4 Coccidiasina; albohirtum) Adelina AF494059 Eucoccidiorida; n/a bambarooniae Adeleorina; Ba3:5 Adeleidae. Adelina grylli DQ096836 Crickets: Gryllus n/a bimaculatus Adelina dimidiata DQ096835 Centipedes: n/a Scolopendra cingulata Klossia helicina HQ224955 Land snails: n/a Clone 2 6 Cepaea nemoralis Klossia helicina HQ224956 n/a Clone 4 3 Cryptosporidium AF115378 Apicomplexa; (Cavia NA wrairi Conoidasida; porcellus) Cryptosporidium AF112574 Coccidiasina; Birds, (Turkeys) meleagridis Cryptosporidiidae Cryptosporidium Felines () felis AF108862 Cryptosporidium AF112571 Mammals (Mouse) parvum Cryptosporidium AF108864 Mammals (Cattle) parvum Cryptosporidium AF108865 Homo sapiens parvum Cryptosporidium sp. AF108860 Koala Kl Phascolarctos cinereus Cryptosporidium sp. AF513227 Eastern grey EGK3 kangaroo. Macropus giganteus Aggregata DQ096837 Apicomplexa; Octopi: Prawns: Palaemon octopiana Conoidasida; vulgaris elegans Aggregata eberthi DQ096838 Coccidiasina; Cutllefish: Prawns: Portunus Aggregatidae officinalis depurator Goussiajanae AY043206 Apicomplexa; n/a Conoidasida; (Leuciscus

75 Coccidiasina; leuciscus) Goussia metchnikovi FJ009244 Eimeriidae Gudgeons: Gobio n/a gobio Goussia noelleri FJ009241 Agile frog (Rana n/a dalmatina) Goussia neglecta FJ009242 Edible frog n/a {Pelophylax kl. esculentus) Goussia sp. Bufo FJ009243 Common toad n/a {Bufo bufo) Choleoeimeria sp. AY043207 Diadem snake n/a {Spalerosophis diademd) Isospora robini AF080612 American robin n/a (Turdus migratorius) Eimeria falciformis AF080614 Mice n/a Eimeria nieschulzi ENU40263 Mice n/a Eimeria acervulina U67115 Chickens n/a Eimeria tenella U67121 Chickens n/a Cyclospora sp. U40261 Humans n/a DQ060674 Apicomplexa; Humans Cystoisospora AF029303 Conoidasida; Canines ohioensis Eucoccidiorida; Cystoisospora suis U97523 Eimeriorina; Pigs Frenkelia glareoli AF009245 Sarcocystidae Clethrionomys glareolus Frenkelia microti AF009244 Microtus arvalis Toxoplasma gondii EF472967 Felines Rodents Neospora sp. BPAl U17345 Canines Cattle Neospora caninum AJ271345 Sarcocystis mucosa AF109679 Carnivorous Marsupials marsupials S. lacertae AY01511 Smooth snake Wall lizard {Coronella {Podacris muralis) austriaca) S. scandinavica EU282032 Canines Moose (Alces alces) S. rangi EF056011 Foxes: Vulpes sp. Reindeer {Rangifer S. grueneri EF056010 tarandus) S. rangiferi EF056016 unknown Sarcocystis sp. AB257157 unknown Sika deer {Cervus HM050622 nippon)

16 mirabilis DQ176427 Apicomplexa; Water scorpions: Conoidasida; Nepa cinerea Gregarinasina; ; Syncystidae sp. AY334569 Apicomplexa; Fire ants: SV-2003 Conoidasida; Solenopsis invicta Mattesia geminata AY334568 Gregarinasina; Fire ants: NA Neogregarinorida; Solenopsis geminata Ascogregarina DQ462454 Apicomplexa; Culicine taiwanensis Conoidasida; mosquitoes Ascogregarina DQ462459 Gregarinasina; armigerei Eugregarinida; Ascogregarina DQ462457 culicis a For ranks above orders, taxonomic positions are based on Adl et al, 2005. Lower level taxonomic ranks are based on Levine, (1988) b For parasitic species, the definitive host is that species in which gametes are formed and syngamy occurs 0 For parasitic species, the intermediate host is a required participant in the life cycle of the parasitic species, but in which only asexual development or replication occurs.

77 CHAPTER IV: CYTOCHROME OXIDASE I: A USEFUL MITOCHONDRIAL GENE FOR DNA BARCODING AND PHYLOGENETICS OF COCCIDIAN PARASITES WITH PARTICULAR REFERENCE TO ElMERIA SPP. (ALVEOLATA, APICOMPLEXA, ElMERIORINA)

(Contents of this chapter have been accepted for publication: Joseph D. Ogedengbe, Robert H. Hanner and John R. Barta. 2011. DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimenorina, Apicomplexa, Alveolata). InternatwnalJournalfor Parasitology (in press) - doi: 10.1016/j.ijpara.2011.03.007.

4.1. Abstract

Partial (-780 bp) mitochondrial cytochrome c oxidase subunit I (COI) and near complete nuclear 18S rDNA (-1780 bp) sequences were directly compared to assess their relative information content as markers for species identification and phylogenetic analysis of coccidian parasites (phylum Apicomplexa). Fifteen new COI partial sequences were obtained using two pairs of new primers from rigorously characterized (sensu Reid and Long, 1979) laboratory strains of 7 Eimeria species infecting chickens as well as 3 additional sequences from cloned laboratory strains of Toxoplasma gondii (ME49 and GT1) and Neospora caninum (NCI) that were used as outgroup taxa for phylogenetic analyses. Phylogenetic analyses based on COI sequences yielded robust support for the monophyly of individual

Eimeria species infecting poultry except for the Eimeria rnitis/mivati clade; however, the lack of a phenotypically characterized strain of Eimeria mivati precludes drawing any firm conclusions regarding this observation. Unlike in the 18S rDNA-based phylogenetic reconstructions, Eimeria necatrix and E. tenella formed monophyletic clades based on partial

COI sequences. A species delimitation test was performed to determine the probability of making a correct identification of an unknown specimen (sequence) based on either complete

18S rDNA or partial COI sequences. In almost all cases, the partial COI sequences were more reliable as species-specific markers than complete 18S rDNA sequences. These observations demonstrate that partial COI sequences provide more synapomorphic characters at the species level than complete 18S rDNA sequences from the same taxa. We conclude that COI

78 performs well as a marker for the identification of coccidian taxa (Eimeriorina) and will make an excellent DNA 'barcode' target for coccidia. The COI locus, in combination with 18S rDNA as an 'anchor', has sufficient phylogenetic signal to assist in the resolution of apparent paraphylies within the coccidia and likely more broadly within the Apicomplexa.

4.2. Introduction

The eimeriorinid coccidia (Eimeriorina, Minchin 1903) consist of several groups of apicomplexan parasites including pathogens of veterinary and medical importance. These include species in the Eimeriidae and Sarcocystidae such as Eimeria spp. and Toxoplasma gondii, respectively. Whether homoxenous or heteroxenous, parasites in these families undergo merogony, gamogony and formation of oocysts in the definitive host that are then shed either sporulated or unsporulated in the feces. Unsporulated oocysts shed in the feces sporulate after leaving the body when exposed to correct environmental conditions of temperature, moisture and oxygen. Members of the family Eimeriidae share distinct morphological features, such as a Stieda body in a variable number of sporocysts that varies with the genus, and refractile bodies within the enclosed sporozoites. This family possesses the largest number of described species within the phylum Apicomplexa (Perkins et al., 2000) that belong to many well described genera including Eimeria, Caryospora, Cyclospora,

Isospora, Tyzerria among others. The number of sporocysts contained in a sporulated oocyst is typically used to assign coccidia to a particular genus within the family Eimeriidae (Perkins et al., 2000). The most speciose (over 1000 species described) of these genera is Eimeria, where oocysts contain four sporocysts that each enclose two sporozoites. Eimeria spp. are an important burden on the health and productivity of domestic animals where the total cost of prophylaxis, chemotherapy and morbidity runs into millions of dollars annually in poultry

79 alone (Dalloul and Lillehoj, 2006). Hence there is a pressing need for rapid, accurate and cost effective identification tools. Oocysts containing four sporozoites in each of two sporocysts are typical of some parasites in both the Eimeriidae and the Sarcocystidae. However, parasites such as Isospora robini and Isospora gryphoni that have a Stieda body in each sporocyst and sporozoites containing refractile bodies have been shown by molecular phylogenetic analyses to belong to the family Eimeriidae (see Carreno and Barta, 1999) whereas morphologically similar monoxenous parasites of mammals that lack Stieda bodies have been transferred to the genus Cystoisospora within the Sarcocystidae (Barta et al., 2005). The identification and classification of eimeriid coccidia has been based largely on morphological and life cycle details (Levine, 1970; 1988). These classifications have been subjected to revisions, based on newly discovered features and more detailed biological studies. More recently, molecular data have been explored in an attempt to explain some rather confusing associations, not just within the Eimeriidae but within the phylum Apicomplexa generally.

Morrison et al. (2004) described the usefulness of 18S rDNA in classifying apicomplexan parasites, especially in support of higher taxonomic groupings within the phylum. Using 18S rDNA sequences from a variety of coccidian parasites, Morrison et al.

(2004) noted that classical taxonomy of the monoxenous coccidia in the family Eimeriidae was not well supported by the molecular data; specifically, sequence data were unable to confirm the monophyly of all of the Eimeria spp. included in the analysis. Coccidia with morphologically distinct oocysts such as Isospora spp. infecting birds and Cyclospora spp. infecting mammals were interspersed among the Eimeria spp. making the latter genus paraphyletic. Similarly, Martynova-VanKley et al. (2008) found that near complete 18S rDNA sequences from chicken Eimeria spp. did not resolve into monophyletic clades.

80 Mitochondrial genes appear to give useful phylogenetic signals in many organisms that use oxidative phosphorylation as a means of respiration (Boore, 1999; Le et al, 2000) and cytochrome c oxidase subunit I (COI) in particular has shown to be useful in delineating recent speciation events (Lane, 2009) and may be a useful adjunct to nuclear genes for inferring recent evolutionary events. The use of genetic data to identify organisms has a long history and is gaining acceptance and popularity as an adjunct to morphology-based identifications. DNA barcoding has emerged as a standardized, sequence-based identification procedure that exploits genetic differences in specific gene regions of eukaryotic organisms, to aid species identification and document their diversity (Hebert et al., 2004a; 2004b).

Mitochondrial COI has been the most widely used genetic target in animal barcoding and has been found to be useful in species delineation on a large scale (Teletchea 2010) with more than 1 million COI sequences having been deposited on the Barcode of Life Data System

(BOLD; Ratnasingham and Hebert, 2007) as of this writing. These developments have given rise to the International Barcode of Life Project (iBOL.org) which is calling for a barcode- based identification system for animals, including parasites. The barcoding technique involves the use of a short (-600-800 bp), standard "PCR friendly" gene region that is of universal occurrence and that can provide sufficient variability needed for distinguishing species efficiently (ie. from a single read using a traditional Sanger sequencing approach).

According to Ferri et al. (2009), a useful DNA barcode gene must exhibit variability between species, should be short enough to be sequenced in a single reaction and contain sufficiently conserved regions to facilitate the development of universal primers to enable PCR amplification of the target marker across as much taxonomic breadth as possible. The variation within species should be smaller than between species for the technique to work

81 reliably. COI has been used alone or in conjunction with other genes in phylogenetic analysis or molecular identification of parasites including Eimeria species (e.g. Ros and Breeuwer,

2007; Huang et al, 2009; Schwarz et al., 2009).

The objectives of this study were to investigate the utility of cytochrome c oxidase subunit I partial sequences in identification and phylogenetic analysis of some parasites in the genus Eimeria, and to compare the phylogenetic informativeness (sensu Roe and Sperling,

2007) of partial COI sequences with the much longer 18S rDNA sequences traditionally used for molecular phylogenetic studies of coccidia. Similarly, the relative utility of mitochondrial

COI gene sequences for species identification of coccidia was compared with that of more commonly used nuclear 18S rDNA sequences.

4.3.1. Oocysts andDNA extraction

For all Eimeria spp. from chickens propagated in our laboratory, single oocyst or single sporocyst strains were isolated from field samples and amplified in specific pathogen free birds. Assignment of each strain to a particular Eimeria species was made based on oocyst measurements, location and histological appearance of endogenous stages and clinical appearance of infection as in the original species descriptions summarized by Reid and Long

(1979). A laboratory strain of the murine parasite Eimeria falciformis was kindly provided by

Dr. Bill Chobotar (Andrews University, MI, USA). For DNA isolations, oocysts were later separated from fecal debris, washed and sporulated in 2.5% potassium dichromate. After sporulation, oocysts were purified and DNA extracted using DNAzol reagent (Invitrogen,

USA) or prepGEM™ Blood kit (ZyGEM, Hamilton, New Zealand) according to the manufacturer's protocol but with the addition of 500nm glass beads (Ferro Microbeads;

Cataphote Division, Jackson Mississippi, USA) to promote the release of sporozoites from

82 sporocysts and oocysts. Samples were vortexed for 30 - 60s to aid in oocyst disruption into the DNAzol or prepGEM reagent. DNA quantities were later estimated using a spectrophotometer (GeneQuant Pro, Amersham Biosciences; Golden Valley, MN, USA) and adjusted to a concentration of ~35ng/ul in the final working dilution.

4.3.2. PCR reaction parameters

PCR was carried out in a MJ mini thermal cycler (Bio Rad, CA, USA) using the following parameters in a 50ul reaction. After an initial denaturing temperature of 96°C for

5min, 40 cycles of 94°C for 20sec, 55°C for 30sec and 72°C for 90sec were run followed by a final extension at 72°C for lOmins. About 70 ng of parasite DNA was used as template for each PCR reaction with one unit of Platinum Taq (Invitrogen, Carlsbad CA, USA), ImM each of Eimeria-spQcific cytochrome oxidase I primers (Cocci_COI_For;

GGTTCAGGTGTTGGTTGGAC and Cocci_COI_Rev; AATCCAATAACCGCACCAAG),

1 xPCR buffer (Invitrogen) and 2.5 mM MgCl2. A similar region of the COI gene was amplified from purified DNA samples obtained from Toxoplasma gondii Strains ME49, T. gondii Strain GT1 and Neospora caninum Strain NC-1 (kindly provided by Dr. J.P. Dubey,

USDA, Beltsville, MD) using Toxoplasmatinae-specific primers (ToxoCOIFor;

GGAGGAGGTGTAGGTTGGAC and Toxo_COI_Rev; CATTTTGTATTATCTCTGGG) with the same PCR reaction conditions as above. Note that metazoan-specific 'universal'

COI primers LCO1490 and HC02198 (see Folmer et al., 1994) designed to amplify -650 bp at the 5'end of the mt COI gene (the BARCODE region) failed to produce amplicons from any of the coccidial DNA templates using the same amplification parameters, necessitating the development of new taxon-specific primers. Both negative and positive controls were included in all PCR reactions. PCR products were later electrophoresed on a 1.5% agarose

83 submarine gel in lx Tris-acetate-EDTA (TAE) buffer at 120V for 45 minutes. The resulting gel was stained with ethidium bromide. PCR products (-800 bp - either in solution or excised from an agarose gel) were later purified using Roche High Pure DNA purification kit

(Roche Applied Science, Germany). Amplicons were sequenced in both directions using

PCR cycle sequencing using the forward or reverse amplification primers followed by detection on ABI Prism 3730 or 3100 DNA sequencers. All newly reported specimen provenance data, related images and electropherogram 'trace' files were deposited in the publicly accessible project ('Eimeria Barcodes' [EIMER]) within BOLD (Ratnasingham and

Hebert, 2007). Data generated for this study generally comply with the BARCODE standards

(Hanner, 2009). However, the new primers developed for Eimeria only exhibited a partial overlap (-328 bp) with the standard BARCODE region of COI.

4.3.3. Sequence Alignment and Phylogenetic Analysis

COI sequences were obtained as optimized contigs using Geneious Bioinformatics software package (Ver. 4.7.6 and subsequent versions), Biomatters Ltd., New Zealand

(Drummond et al., 2010). Each new COI sequence was approximately 800 base pairs in length. Newly generated and existing COI sequences from GenBank were aligned using

Clustal-X (Larkin et al., 2007) and translation-based alignment implemented from within

Geneious. The resulting alignment used in these analyses was 782bp in length including gaps but with primer regions trimmed. The protein translation of the sequences presented open reading frames without stop codons and there was no obvious codon bias when the sequences were analyzed using MEGA version 4 (Tamura et al., 2007).

Phylogenetic analysis described in detail below was performed on 109 mt COI sequences; 106 sequences representing 9 Eimeria species from various hosts formed the ingroup for analysis

84 and 3 sequences from members of the Toxoplasmatinae {Toxoplasma gondii Strains GT1 and

ME49, and Neospora caninum Strain NC-1) were used as taxonomic and functional outgroups. The newly generated mt COI sequences (followed by GenBank Accession number) used in the analyses followed by pre-existing sequences downloaded from GenBank were: E. acervulina Strain NC3 (HM771673) and Strain USDA 84 (HM771674) plus

EF158855, FJ236428, FJ236427, FJ236420, FJ236419, FJ236443; E. brunetti Strain

Guelph 80 (HM771675); E. mitis Strain USDA 50 (HM771681); E. mivati FJ236433,

FJ236441, FJ236434, EF174185; E. maxima Strain M6 (HM771684), Strain Guelph 74

(HM771685) and Strain USDA 68 (HM771686), plus EF174183, EF174184, EU025104-

EU025106, FJ236380, FJ236386-FJ236394, FJ236401, FJ236402, FJ236406-FJ236418,

FJ236429.FJ236432, FJ236435-FJ236440, FJ236442, FJ236448-FJ236452, FJ236454,

FJ236456, FJ236457, FJ236459, EU025107,;£. necatrix Strain Guelph 84 (HM771680) plus EU025108; E. tenella Strain USDA80 (HM771676), Strain MD1 (HM771677), Strain

Guelph ID (HM771678) and Strain Guelph 2D (HM771679) plus EF174186-EF174188,

EU025109, FJ236381-FJ236384, FJ236396, FJ236395, FJ236397-FJ236399, FJ236385.

FJ236400, FJ236403-FJ236405, FJ236421-FJ236426, FJ236430, FJ236444-FJ236447,

FJ236453. FJ236455, FJ236458, E. falciformis Strain Chob2 (HM771682); E. zuernii Strain

Guelph 2007 (HM771687); Neospora caninum Strain NCI (HM771688); Toxoplasma gondii

Strain ME49 (HM771690); T. gondii Strain GT1 (HM771689).

Phylogenetic analysis described in detail below was performed on 106 nu 18S rDNA sequences; 94 sequences representing 8 Eimeria species from chickens formed the ingroup for analysis and 12 sequences from members of the Toxoplasmatinae were used as taxonomic and functional outgroups. Sequences were aligned initially using Clustal-X implemented from

85 within Geneious, followed by refinement by eye using a staggered alignment method to maximize positional homology (Barta 1997). The nu 18S rDNA sequences (followed by

GenBank Accession number) used in the analyses were: E. acervulina (EF210323.1,

EF210324.1. EF175928.1. DQS38351.1. DQ136187.1, FJ236372.1, U67115.1); E. brunetti

(U67116.1); E. maxima (FJ236329.1, FJ236333.1, DO538350.1. DQ640012.1, DQ136186.1.

EU025110.1, FJ236332.1, FJ236330.1. FJ236331.1, EU025111.1, FJ236334.1,

EU025112.1. FJ236335.1, FJ263947.1, FJ236336.1, FJ236360.1, EF122251.2.

DQ538349.1, FJ236339.1, FJ236355.1, FJ236353.1, FJ236346.1, FJ236347.1, FJ236348.1,

FJ236344.1, FJ236354.1, FJ236345.1, FJ236358.1, FJ236359.1, FJ236338.1. FJ236343.1,

FJ236350.1. FJ236352.1, FJ236342.1, FJ236351.1, EF210322.1, FJ236357.1, FJ236341.1,

FJ236349.1. U67117.1, DQ538348.1, FJ236361.1, FJ236356.1, FJ236340.1, FJ236337.1);

E. mitis (TJ40262.1, U67118.1, FJ236379.1); E. cf. mivati (FJ236373J., FJ236376.1,

FJ236375.L FJ236378.1, FJ236374.1, FJ236377.1); E. mivati (U76748); E. praecox

(FJ236362.1, FJ236367.1, FJ236366.1, FJ236368.1, FJ236370.1), FJ236363.1,

FJ236365.1. GQ421692.1, FJ236364.1, FJ236371.1, FJ236369.1, U67120.1; E. necatrix

(DQ136185.1, U67119.1); E. tenella OJ67121.1, EU025114.1, EF210325.1. DQ640011.1,

EU025116.1, AF026388.1, DQ136180.1, DQ136176.1. U40264.1, DQ136182.1.

EU025113.1. DQ136184.1, DQ136179.1, DQ136177.1, DQ136181.1, EU025115.1,

DQ136178.1. DQ136183.1); Eimeria falciformis (AF080614); Eimeria zuernii (AY876932);

Neospora caninum (U03069.1, AJ271354.1, U17346.1, L24380.1); Neospora sp.

(TJ17345.1), Toxoplasma gondii, (U00458.1; M97703.1; U03070.1; EF472967.1; U12138.1;

L37415.1; L24381.1).

86 Nucleotide evolutionary models were evaluated using MrModeltest 3.7 (Nylander,

2004) in PAUP 4.0M0 (Swofford, 2003). The best-fit model, according to the Akaike

Information Criterion evaluation of hierarchical likelihood ratio tests suggested the general time reversible model with gamma (GTR+G) distribution of nucleotide substitution for all analyses. In all analyses, the two T. gondii strains and N. caninum were used as the taxonomic outgroup. Both maximum likelihood and maximum parsimony methods were carried out using nucleotide substitution parameters generated using MrModeltest 3.7

(Nylander, 2004). The suggested Bayesian parameters for phylogenetic tree estimation were added to the dataset and the Markov Chain Monte Carlo performed at 5 million generations, with 4 chains, 2 runs and a sampling frequency of 1000 (Huelsenbeck and Ronquist, 2001;

Ronquist and Huelsenbeck, 2003). The 'burnin' value was set at 100,000 and the resulting consensus tree was examined using Treeview-Win 32 (Page, 1996).

4.3.3. Species delimitation using mt COI or nu 18S rDNA sequences

The relative utility of mt COI or nu 18S rDNA sequences for the delimitation and identification of a number of Eimeria species was assessed using the Species Delimitation plug-in (Masters et al., 2011) within Geneious (Drummond et al., 2010). This application measures the intraspecific and interspecific genetic differences and then uses the method of

Ross et al. (2008) to estimate the probability that a new sequence would be correctly assigned to a putative species using the current multiple sequence alignment and tree as the reference dataset. Comparisons were made for seven of the Eimeria species that infect chickens for which both nu 18S rDNA and mt COI sequences were available. Because the BOLD

'identification engine' (Ratnasingham and Hebert, 2007) includes only 5' COI sequences, it

87 could not be used to perform a species identification analysis with the legacy data involving sequence data outside the barcode region.

4.4. Results

Figure 4.1 illustrates the consensus trees generated for COI and 18S rDNAusing

MrBayes and estimates of nucleotide substitution by MrModeltest (Nylander, 2004). Both maximum parsimony and maximum likelihood trees showed similar topologies and all three methods generated trees that showed support for the monophyly of each chicken Eimeria spp.

(MP & ML trees not shown). The strict consensus tree generated by maximum parsimony from the mt COI sequences had a tree length of 718 steps with a consistency index of 0.708.

Of the 782 aligned nucleotide positions, there were 386 constant characters, 116 variable characters that were parsimony uninformative and 280 variable characters that were parsimony informative. The maximum parsimony consensus tree for the nu 18S rDNA was

682 steps in length with a consistency index of 0.73; 1347 characters were constant, 145 variable characters were parsimony uninformative and 287 variable characters were parsimony informative.

88 Figure 4.1. Phylogenetic trees based on Bayesian analyses of nuclear 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences. Maximum likelihood and maximum parsimony trees were similar to the Bayesian trees for both genes (data not shown). Horizontal distance is proportional to hypothesized evolutionary change as indicated (scale bar). The posterior probability branch support is shown. In both analyses, support for the monophyly of individual Eimeria species infecting poultry was strong except for the Eimeria mitis/mivati clade. A. Consensus tree for nuclear 18S rDNA sequences of Eimeria species infecting poultry using members of the Toxoplasmatinae as outgroup taxa. B. Consensus tree for mitochondrial cytochrome c oxidase subunit I (COI) sequences of Eimeria species using members of the Toxoplasmatinae as outgroup taxa. Note: All sequences generated in our laboratory from phenotypically characterized laboratory strains of parasites are underlined; GenBank accession numbers are bolded for newly generated sequences; 18S rDNA and COI sequences generated by Schwarz et al. (2009) were obtained from 4 pooled DNA samples of oocysts (AL/L, AL/H, NC/L and NC/H as indicated, each containing 4-5 mixed Eimeria spp.) and assigned to species based on BLAST similarity (see text for further details); all other sequences were obtained directly from GenBank and were assigned to species by the contributing authors.

89 A-Nuclear 18S rDNA B - Mitochondrial COI

j- Eimeria acervulma EF'210323 J5raemgcen'ufr»aUSDA84 HM77I674 A_| Bimercaac emilijiaEF210324 Eimeria acervulma NC 3 HM77167 3 U°l fimerfaaceraill>iaEF175928 Eimeria acervulma EF158855 f Eimeria acervulma U67115 Eimeria acerwhna NC/H FJ236427 I Bmena acera/linaDQ538351 • Eimeriaacemilmanaa FJ236420 \ Eimeria acervuhnaT>Q\%W 1 EimenaacemilinaNCA* FJ236443 *• Enmria acervulma NC/L FJ236372 p Eimeria acervulma NC/H FJ236428 Kmer/amM* U76748 ^-Eme//aacemjJz)iaNC/HFJ236419 Eimeria cf mivtrtiNC/H FJ236378 Etmeriabrunetti GuelT3h80 HM771675 Bmena cf rain* N C/H FJ236377 9$p fimejia cf mvati NC/H FJ236441 Bmena cf mtvati NC/H FJ236373 amenacf imwo(iNC/HFJ236433 Bmexiacf minrfiNC/HFJ236376 100 Elmertamith USDA 50HM771/J81 Eimeria cf mivafl NC/H FJ236375 100[j flraerto cf ra/vtii/ NC/H FJ236434 Bmxna cf mivati NC/H FJ236374 SmeriaminrfiEF174185 Eimeriamitis NC/HFJ236379 lonl-J fimertamms U40262 Simena raox/mo NC/H FJ236432 '""—1 BmxnamtaUGim Smena manmaNC/HFJ236440 Eimeria praecox AR/L FJ236362 97 Bmer/amaxfmaM6 HM77r-_. - Bmena praecox AR/L FJ236363 Eimeriamaxima Unelph /4HCT771685 Eimeria praecox ARL FJ236365 gimenamaKraaUSDh68HM771686 Eimeriamaxima AK/L FJ23641U 100 100 ' Eimeria praecox U67120 Eimeria praecox ARIL FJ236366 ameTO manmaNC/H FJ236436 • Eimeria praecox AR/L FJ236364 Eimeriamaxima AR/L FJ236414 •• Eimeria praecox AR/L FJ236367 Eimeriamaxima EU025106 J- Eirnerta praecox GQ421692 05 Eimeriamaxima EU025105 r Eimeria praecox AR/L FJ236369 S/m8i'/amai(maEU025104 f- Bmena/iraeroi AR/L FJ236368 £/menamax/maEF174184 L- Eimeria praecox AR/L FJ236370 Eimeria maximum EF174183 *• Eimenapraecox AR/L FJ236371 Sm«rtamaxfmaEU025107 —"" Bimenaininetftl^?!^ Eimeria maxima AR/L FJ236413 100 • fliw/a max/ma AR/H FJ236387 99 Eimeria maxima EU025112 Smer/araax/maNC/L FJ236457 p Bmena maxima AR/L FJ236329 Smsj-iaraax/maNC/L FJ236452 1 Brasi-ja maxima AR/L FJ236333 amerjamai/ma AR/L FJ236411 • Eimeria maxima AR/L FJ236332 Eimeriamaxima NC/L FJ236459 Eimeriamaxima NC/L FJ236331 Eimeriamaxima NC/L FJ236454 Eimeria maxima DQ136186 Bmerfa max/ma NC/L FJ236450 Bmena waxmv? AR/L FJ236330 flmei-iamar/raaNC/H FJ236439 I Emma maxima DQ538350 Eimeria maxima AR/L FJ236408 96 * Eimeria maxima EU025110 Eimeria maxima AR/H FJ236380 I" Eimeria maximaEU025111 Eimeria maxima AR/L FJ236409 I r BmenamaxlmaEF122251 Eimeriamaxima NC/L FJ236456 I Bmena maxima DQ538349 Bmer/a max/ma NC/L FJ236451 I Jfime«amax/maDQ640012 fimeiva max/maNC/L FJ236448 ^ I*-Bmena maxima NC/L FJ236339 Bmena maximaNC/H FJ236437 Eimeria maxima AR/L FJ236360 Sme«a max/ma AR/L FJ236418 Bmeria maxima AR/L FJ236412 L Bmerm maxima DQ538348 100 « Smeria mcraraa AR/H FJ236390 r Bmena maximaNC/LFJ236359 L" BmenamoximaNC/L FJ236341 • Bmei'/a max/ma NC/H FJ236435 r Bmena maxima EF210322 • Bmer/a wax/ma AR/H FJ236393 I I- Eimeria maxima NC/L FJ236340 Bmeria max/ma AR/H FJ236388 Smena maxima NC/H FJ236355 • Bmerra maxima NC/L FJ236449 Braerra maxima NC/H FJ236357 1 Eimeria maxima NC/L FJ236442 Enneriamaxima ARIL FJ236353 fflnw/araax/raa ARH FJ236386 Eimeriamaxima AR/L FJ236354 Bmena maxima AR/L FJ236416 • Eimeria moamaNC/L FJ236350 Bitwia maxima NC/H FJ236438 • Bmex/amaximaNC/L FJ236352 • Bmena maxima ARH FJ236391 Bmena maximaNC/L FJ236351 Eimeriamaxima AR/L FJ236415 Bmena maxima NC/H FJ236356 Bmei'ia maxima ARH FJ236392 Bmena maxima AR/L FJ236343 flmona maxima NC/H FJ236429 Eimeria moximaNC/H FJ236348 K Bmena maxima AR/L FJ236402 Eimeria maxima AR/H FJ236349 ajmra max/maNC/H FJ236342 1 Eimeriamaxima AR/L FJ236417 Eimeria moximaNC/H FJ236344 u- Bmeria maxima AR/L FJ236407 Eimeriamaxima AR/L FJ236346 t flmexiamaama AR/L FJ236406 Emeriamaxima AR/L FJ236345 . Bimei-ia maxima AR/L FJ236401 r Eimeria maxima N C/L F1236361 ^ Bmena maxima AR/H FJ236389 tr- BmenamaximaNC/HFJ236338 12Pj,amenaii8i:afrixEU025108 P1 Braer/ a maxima U 67117 ~ flmeria necafrix Guelnli 84 HM771680 U- Eimeria moximaNC/L FJ236336 r Emena tenella USDA80 HM771676 T— Eimeria maxima NC/L FJ263947 • flimgria tenella MP 1 HM771677 I— Eimeria maxima AR/H FJ236337 y Eimeria tenella Guelnh ID HM771678 Eimeriamaxima NC/H FJ236358 'Ji'imcl-iafae»aGuelllh2DHIvl//167V Bim2xiatoe«aEU025109 fi/mer/a maxImaNOL FJ236335 Bmei-iaten8HaEF174188 Bmena tenellaDQ136180 Bimenate>rcHaEF174187 Eimeria tenella DQ136176 •Eimeria (endiaEF174186 Eimeria temlla DQ136182 • Eimeria tenella AR/L FJ236404 Bmertate>!eMaEU025115 100 fimercatejiefia NC/L FJ236446 Bmerta tenella DQ136179 Eimeria tenellaNCIL FJ236444 Eimeria tms.Ha DQ 136177 Bmena toeHaNC/L FJ236458 0 025 100 • Bmena tenellaDQ136178 Eimeria tenella NC/L FJ236453 Bmena fcneWaEF210325 • BmenatorcHaNC/L FJ236447 Bmena teellaEU025116 Bmenate«WaNC/H FJ236423 Eimenatenella AF026388 Bmsriatae/teNC/H FJ236422 KL Bmena a temlla DQ136181 Bmma lem«aNC/H FJ236421 I Eimeria tenellaEU025114 Bmsna tenellaN OH FJ236430 LBmenateneflaU67121 Eimeria tenella NC/H FJ236424 |" Eimenatenella U40264 • Bmsi'ia tenella AR/H FJ236400 II Eimeria tenella EU025113 • Emeriatenella AR/H FJ236396 P" Eimeria tenella DQ640011 Ermeriatenelta AR/H FJ236395 b flmeiva tenella DQ136184 • Eimenatenella AR/H FJ236384 P" Eimeria tenella DQ 136183 10C • amenatenella AR/H FJ236381 , nrl_J Eimeria necatrix DQ 136185 SiwrartoKlto NC/L FJ236445 1 «^T- £,mena necatrix U67119 Eimertatenella NC/L FJ236455 —— flmena/aJcijf>mls Ctlob2 AF080614 Eimeria tenella AR/L FJ236405 — Eimerianiernll AY876932 Bma-iateneJJaNC/H FJ236426 ^ roio^teraafona';iU00458ts4 Bmena tene fla NC/H FJ236425 roro;teraagona';/U12138 S48 Bmenafene/laAR/L FJ236403 • Toxoplasma gondii EF472967 RH Bmeriatene/laAR/H FJ236399 ' Toxoplasma go«a// M97703 RH flmeriatenetta AR/H FJ236398 ^^ Toxo/i/asmagona';/L24381 Eimenatenella AR/H FJ236397 ^T^T | Toxoplasma gondii L37415 iuu , • Bimena tenella AR/H FJ236385 7bxo^lasraago«Ji;U03070 Bmena temlla AR/H FJ236383 r Neospora camnum U03069 Eimenatenella AR/H FJ236382 |Afeoi/)o/'aca«/«ijfflAJ27t354 Eimarta;iiei7iilG«lph2007HM771687 I Ikospora canimim U17346 Eirmrlazuemil Gue ph2007 89IA6os/>ora sp U17345BPA1 —^Eimeria taicilormis Choo2 HM77168: ^Neosporacaninum L24380 j^i^» Neospora camnum MCI HM771688 i__| Toxoplasma gondii Strain GT HM771689 100' ' Toxoplasma gondii ME49HM771690

90 Table 4.1 illustrates the pairwise estimates of sequence divergence between the parasites included in this study. Table 4.2 demonstrates intraspecific and interspecific sequence diversity of parasites for which more than one sequence of a particular genetic target was available. The genetic distances between the different taxa in both the mt COI and nu 18S rDNA sequences were computed using 109 sequences for cytochrome oxidase I and 106 sequences for the 18S rDNA.. The aligned lengths (including alignment gaps) of the mt COI sequences was 782 bp compared with 1778 bp for the 18S rDNA sequences. Nonetheless, the number of phylogenetically informative characters for each alignment was almost the same

(280 for the COI alignment versus 287 for the 18S rDNA alignment). This was reflected in the pairwise distances of mt COI sequences; variation between Eimeria species ranged from

0.011-0.17 (Table 4.1) whereas variation within a single Eimeria sp. was limited (0.00023-

0.004) (Table 4.2). The largest variation between Eimeria species infecting birds was between

E. maxima and E. necatrix (0.17), which was the same as between E. maxima and E. zuernii of cattle (data not shown). Mean pairwise genetic distances between species using nu 18S rDNA sequences indicated markedly lower genetic variation. For example, among the various

Eimeria spp. infecting chickens, the greatest genetic distance between species was

0.042±0.006 for nu 18S rDNA sequences compared to 0.171±0.038 for mt COI sequences

(Table 4.1).

Within the Eimeria species infecting chickens, intraspecific variation in the mt COI sequences (0.00023-0.004) and nu 18S rDNA sequences (0.002-0.013) was similar (Table

4.2). However, as noted above, interspecific genetic distances from an Eimeria species to its nearest genetic relative were generally greater for the mt COI sequences (0.020-0.098) compared with the nu 18S rDNA sequences (0.009-0.027). The similar intraspecific distances

91 but larger interspecific distances with the mt COI sequences were reflected in the lower intraspecific/interspecific ratios for that genetic target. These greater genetic distances between mt COI sequences from Eimeria species of chickens produced higher P ID(Liberal) values (Table 4.2 - Panel A), when compared with P ID(Liberal) values obtained for nu 18S rDNA sequences (Table 4.2 - Panel B). A higher P ID(Liberal) value serves as a prediction of the utility of a genetic target for species delimitation (Ross et al., 2008; Masters et al., 2011).

As an example, with both mt COI and nu 18S rDNA sequences, E. tenella was the closest genetic relative of E. necatrix; however the P ID(Liberal) value (plus 95% confidence interval) for mt COI was 0.98 (0.82, 1.00) whereas the P ID(Liberal) value for nu 18S rDNA sequences was only 0.77 (0.62, 0.93).

4.5. Discussion

Eimeria tenella has recently been shown to possess a 6.2kB concatenated mitochondrial genome (found at a 50-fold abundance relative to the nuclear genome of these parasites) that possesses 3 protein coding genes (cytochrome c oxidase subunits I [COI] and

III [COIII], cytochrome b[cytB]) plus 19 large and small subunit rRNA gene fragments

(Hikosaka et al., 2010). The genetically similar mitochondrion of Plasmodium spp. (see

Hikosaka et al., 2010) has been shown to be inherited uniparentally from macrogametes

(Wilson and Williamson, 1997). Perkins and Schall (2002) demonstrated that mt cytB sequences were highly useful for inferring phylogenetic relationships among haemosporidian parasites, yet cytB has not been advanced as a standard marker for species identification for parasites.

The utility of mt COI sequences for species identification and phylogeny reconstruction has been demonstrated for several parasite groups (Pages et al., 2009; Alcaide

92 et al., 2009; Huang et al, 2009). Martinsen et al. (2008) used mt COI sequences in addition to both plastid and nuclear genes to study the evolutionary relationships and events leading to host switching and diversification in Plasmodium species of birds, mammals and squamate reptiles. Cunha et al. (2009) have also used cytochrome c oxidase gene sequences (COI and

COIII) in the molecular epidemiology of Plasmodium by developing highly sensitive (100%) and specific (88%) PCR-based methods for the diagnosis of P. vivax and P. falciparum.

However, adoption of barcode meta data standards (Hanner, 2009) such as the reporting of electropherogram 'trace' files are still generally lacking. In the present study, we examined the utility of the cytochrome c oxidase subunit I (COI) gene for species identification (so- called 'DNA barcoding') and for phylogenetic reconstructions compared with 18S rDNA data of some coccidian parasites, particularly Eimeria species. Importantly, we also deposited standard barcode meta data (trace files, images, catalog numbers of voucher specimens) on

BOLD to enhance the downstream utility of our data for subsequent molecular diagnostic applications.

4.5.1. DNA Barcoding for Parasite Species Identification

The Barcode of Life (BOL) initiative is a global effort to facilitate species identification and discovery using PCR, sequencing and analysis of short standardized DNA sequences known as "DNA barcodes" derived from the 5' region of the mitochondrial COI gene. In the

Apicomplexa, especially the coccidia, there are relatively few studies that have demonstrated that mitochondrial genes could act as useful tools in molecular systematics and/or parasite identification. There are, however, a growing number of workers exploring the mitochondrial genome as a potential tool to address problems in epidemiology, evolution or species identification that could not be solved by relying on the more commonly used nuclear genes

93 alone. For example, Schwarz et al. (2009) used both the ribosomal small subunit gene and mt

COI in assessing the population dynamics of different Eimeria species from various poultry farms. Ferri et al. (2009) applied barcodes in parasitic nematodes, showing high concordance between moprhological and molecular data and because of the ease of analysis stemming from the use of a coding region over a ribosomal marker, made the plea for using COI barcodes over 12S sequences. However, barcoding parasites require development of new primers for reliable PCR amplification (Moszczynska et al. 2009).

In the present study, the utility of 800 bp mt COI sequences was compared to the utility of complete nu 18S rDNA sequences (the most widely available genetic marker for these parasites) for species identification and delimitation. Both newly generated and published sequences used for phylogenetic tree constructions indicate that mt COI sequences can differentiate common species of coccidia in chickens reliably; all resulting clusters of closely related sequences were monophyletic for a single Eimeria species with the exception of the

E. mivati-E. mitis clade. Although two well-supported sister groups were identified within this monophyletic clade, the naming (identification) of the parasites within these sister groups is questionable. Moreover, COI is a protein coding gene and is therefore more straightforward to align, increasing the likelihood that assertions of positional homology in the alignment are correct. All COI sequences generated in our study and many of the 18S rDNA and COI sequences from GenBank were obtained from laboratory strains that were purified and identified as a particular Eimeria species based on observed phenotype (Reid and Long, 1979) prior to DNA isolation, PCR and sequencing. In contrast, Schwarz et al. (2009) assigned mt

COI and nu 18S rDNA sequences obtained from 4 pooled DNA samples of oocysts (each containing 4-5 mixed Eimeria spp.) to species based on BLAST (Altschul et al, 1990)

94 similarity to existing 18S rDNA or COI sequences in GenBank; thus, the species assignments of the resulting sequences cannot be linked to a particular Eimeria sp. phenotype and must be considered tentative (see Fig. 4.1), as is often the case with GenBank data where sequences are reported without reference to specimens examined from reference collections (Ruedas et al. 2000). In all cases where the nu 18S rDNA or mt COI sequences were obtained from a particular Eimeria species that had been identified based on phenotype, the sequences were found within the same clade in each analysis. All sequences obtained by Schwarz et al. (2009) clustered with the sequences from phenotypically characterized Eimeria species thus providing larger sample sizes for each species in our subsequent species delimitation analysis using these data. The genetic distance matrix obtained from 18S rDNA sequences suggests a lower resolution of genetic differences between species when compared with mt COI sequences. Except for sequences from E. maxima isolates, that showed less intraspecific genetic variability in its 18S rDNA sequences than its mt COI sequences (resulting from a single divergent sequence - FJ236432), all other species used in this study showed appreciably greater genetic distances between species based on COI sequences rather than

18S rDNA sequences suggesting COI to be a more useful locus for species delimitation.

These observations suggest that mt COI sequences would be useful for molecular species identification and delimitation for coccidia and, based on similar observations for malarial parasites (Martinsen et al, 2008), perhaps apicomplexan parasites generally. Given sufficient sampling to generate a library of mt COI sequences from classically characterized coccidian species, it should be possible to develop primers and species-specific probes to generate quantitative PCR tools for studying the molecular epidemiology of Eimeria species and other apicomplexan parasites. This would be especially useful in animal hosts where this

95 information could assist in the control of these economically important parasites. However, the importance of using expert-identified reference material is crucial and in this respect our data follow the enhanced annotation and reporting standards associated with BARCODE records in GenBank (as recorded on BOLD). Despite having only partial overlap (-328 bp of the -800 bp PCR product) with the 5' COI BARCODE region for animals, our newly contributed data exceed the minimal length specification (500 bp) and sequence quality score required for a BARCODE record. In sliding window analysis of a number dipteran and lepidopteran COI sequences, Roe and Sperling (2007) demonstrated that the 5'COI DNA barcoding region was no better than other regions downstream in COI; the lengths of the mt

COI sequences obtained were found to be most important for increasing the probability of sampling regions of high phylogenetic informativeness (Roe and Sperling, 2007). The enhanced meta data reporting standard is a key difference between properly conducted DNA barcoding studies and previous molecular assessments, which makes barcode data fit for use in molecular diagnostic applications (Teletchea, 2010).

4.5.2. Use ofmt COI partial sequences for molecular phylogenetics

Both the 18S rDNA and COI-based phylogenetic reconstructions in the present paper were in general agreement with earlier molecular analyses of the relationships among Eimeria species infecting the domestic fowl using 18S rDNA sequences (Barta et al, 1997).

Furthermore, the 18S rDNA- and COI-based reconstructions were largely consistent with one another demonstrating similar branching orders and phylogenetic relationships between and within individual clades. For example, in both analyses, E. mivati and E. mitis sequences clustered together in a monophyletic clade with high bootstrap support, but the monophyly of

E. mivati and E. mitis (using the GenBank species identifications) within that clade was not

96 supported. In the COI analysis, E. brunetti was the sister taxon to a clade containing E. mitislE.mivati and E. maxima; in contrast, E. brunetti was the sister taxon to a large, monophyletic E. maxima clade based on 18S rDNA sequences. However, the lack of any sequences from Eimeria praecox in the COI-based reconstruction may be a confounding factor contributing to the observed branching order differences. Both analyses showed that E. tenella and E. necatrix together formed a well-supported monophyletic clade but only the COI analysis was able to resolve monophyly of E. tenella. The various phylogenetic hypotheses erected based on nu 18S rDNA sequences of Eimeria species from chickens and other hosts suggest that nu 18S rDNA sequences are useful in phylogenetic analyses perhaps only to the genus level. Nuclear 18S rDNA sequences have been unable to resolve the monophyly of

Eimeria species in this and other studies (e.g. Morrison et al, 2004). Whether this reflects a lack of resolving power of this molecular marker or reflects confused taxonomic assignment to a truly paraphyletic genus Eimeria is not known, however recent evidence suggests strongly that genus Eimeria itself is paraphyletic (e.g. Jirku et al., 2009 and Fig. 5.1). Our results support prior assertions (Besansky et al. 2003) that COI could be a widely useful molecular marker with potential for resolving evolutionary relationships among closely related parasites, such as members of the Apicomplexa, that possess a mitochondrion and, in particular, among the morphologically similar coccidia. This marker is likely to provide many species-level synapomorphies that would be highly complementary to analyses based on 18S rDNA sequences. Whether obtained for DNA barcoding, molecular identification or evolutionary studies, DNA sequence datasets should be subjected to robust phylogenetic analyses, such as Bayesian and ML methods with well-aligned sequences and character-based evolutionary models. Phylogenetically appropriate analyses are particularly important for

97 taxonomic groups in which there is only sparse taxon sampling. Such rigorous analyses will address misgivings expressed by DeSalle et al. (2005) that DNA barcode data had been analyzed initially using only distance measures (e.g. Hebert et al. 2004a; 2004b).

4.5.3. Concluding Remarks

DNA barcoding as practiced using phenetic methods is a useful approach for clustering specimens and looking for patterns of genetic discontinuity suggestive of reproductive isolation (e.g. alpha taxonomy). When coupled with robust phylogenetic methods, and likely with additional molecular and/or morphological characters, barcode data can also contribute significantly toward an understanding of phylogeny. For barcoding, rRNA genes could be used to screen samples of completely unknown taxonomy, after which appropriate COI primers could be selected to obtain species-level identifications

(Moczczynska et. al, 2009). Ultimately, these analyses can provide diagnostic characters for subsequent identification of unknown specimens thereby extending the application of taxonomy to epidemiology and diagnostics. Our results clearly suggest that rigorous phylogenetic analyses expand the utility of the COI sequence data beyond just DNA barcoding, at least for the coccidia. Mitochondrial COI sequences have been used successfully to delineate cryptic species of important disease vectors such as Culicoides species (Cunha et al., 2009; Pages et al., 2009) and their parasites (e.g. Haemoproteus or Plasmodium spp.).

This use of mt COI as a marker in the epidemiological study of vector-host interactions as well as our observations suggest a promising future for the mt COI locus as an integral component of DNA barcoding, species identification/delimitation and phylogenetic investigations in the phylum Apicomplexa.

98 TABLE 4.1. COMPARISON OF PAIRWISE DIFFERENCES (MEAN±STANDARD ERROR) OF 18S NUCLEAR RDNA SEQUENCES (UPPER BLOCK) AND MITOCHONDRIAL CYTOCHROME OXIDASE C SUBUNIT 1 (COI) SEQUENCES (LOWER BLOCK) BETWEEN SELECTED COCCIDIAN TAXA.

Eimeria Eimeria Eimeria Eimeria Eimeria Eimeria Eimeria Toxoplasma acervulina brunetti mitis mivati maxima necatrix tenella gondii Eimeria acervulina 0.024±0.004 0.023±0.004 0.016±0.003 0.029±0.004 0.029±0.004 0.027±0.004 0.127±0.013

Eimeria brunetti "o"o99±O.OlVl t 0.025±0.004 0.021±0.004 0.027±0.004 0.040±0.005 0 038±0.005 0.136±0.014 i -, « % - % Eimeria mitis 0.106±0.020 "{no2±b!o2o""" 1 0.014±0.003 0.033±0.004 0.039±0 005 0.035±0.005 0.138±0.014

v Eimeria mivati 0.109±0.020 0.103±0.019 o.on±broo3" 0.030±0.004 0.032±0.004 0.030±0.004 0.134±0.014 1 ? Eimeria maxima 0.139±0.025 0.144±0.026 0.129±0.023 (U22±0.021"""[ 0.042±0.005 0.039±0.005 0.125±0.013

Eimeria necatrix 0.101±0.018 0.124±0.022 0.136±0.024 0.140±0.024 0.170±0.030^| * ~ 0.008±0.002 0.128±0.013

Eimeria tenella 0.104±0.019 0.124±0.022 0.129±0.023 0.131±0.023 0.163±0.028 0.018±0.005 j 4 0.126±0.013 \ 4 Toxoplasma gondii 0 354±0.086 0.342±0.082 0.356±0.088 0.365±0.090 0.345±0.083 0 336±0.082 o.33±b'.o8rr r ''

99 TABLE 4.2. COMPARISON OF THE GENETIC VARIATION WITHIN SPECIES (INTRASPECIFIC DISTANCE) AND BETWEEN CLOSEST SPECIES (INTERSPECIFIC DISTANCE) OF SEVEN ELMERLA SPECIES INFECTING CHICKENS USING EITHER MITOCHONDRIAL CYTOCHROME C OXIDASE - SUBUNIT I (COI) SEQUENCES (PANEL A) OR NUCLEAR 18S RDNA SEQUENCES (PANEL B). THE PROBABILITY (PLUS 95% CONFIDENCE INTERVAL) OF MAKING A CORRECT IDENTIFICATION OF AN UNKNOWN SPECIMEN BASED ON EITHER GENETIC TARGET IS INDICATED BY P ID (LIBERAL).

A. Mitochondrial Cytochrome c oxidase - subunit I (COI) sequences - Species Delimitation

Closest Intraspecific Interspecific Intra/Inter Species P ID(Liberal) Species Distance Distance Ratio (Closest Taxon) E. acervulinoi E. mivati 0.004 0.095 0.04 0.99(0.93, 1.0) E. brunetti E. acervulina n/aa 0.098 0.00 0.96(0.83, 1.0) E. maxima E. mivati 0.004 0.098 0.04 1.0(0.97,1.0) E. mitis E. mivati 0.004 0.033 0.13 0.95(0.84, 1.0) E. mivati E. mitis n/aa 0.033 0.00 0.96(0.83, 1.0) E. necatrix E. tenella 0.00023 0.020 0.01 0.98(0.82, 1.0) E. tenella E. necatrix 0.003 0.020 0.14 0.98(0.95,1.0)

B. Nuclear 18S rDNA sequences- Species Delimitation

Closest Intraspecific „. ? Intra/Inter b Species . T»- * Distance „ . P ID(Liberal) Speciec s Distance ._,, . _, , Ratio _ (Closest Taxon) E. acervulina E. praecox 0.002 0.021 0.11 0.97 (0.91, 1.0) E. brunetti E. praecox n/aa 0.023 0.00 0.96(0.83, 1.0) E. maxima E. brunetti 0.013 0.027 0.47 0.96 (0.93, 0.99) E. mitis E. acervulina 0.005 0.022 0.21 0.89(0.74,1.0) E. mivati E. mitis 0.003 0.014 0.22 0.95(0.85, 1.0) E. necatrix E. tenella 0.003 0.009 0.34 0.77 (0.62, 0.93) E. tenella E. necatrix 0.002 0.009 0.23 0.97(0.94, 1.0)

a Not Applicable: Intraspecific variation uninformative because there is only a single sequence for that species. b P ID(Liberal) The mean probability, with the 95% confidence interval (CI) for the prediction, of making a correct identification of an unknown specimen of the focal species using BLAST (best sequence alignment), DNA Barcoding (closest genetic distance) or placement on a tree, with the criterion that it falls sister to or within a monophyletic species clade (Ross et al., 2008; Masters et al., 2011).

100 CHAPTER V: MOLECULAR PHYLOGENETICS OF EIMERIID COCCIDIA (EIMERIDAE, EMERIORINA, APICOMPLEXA, ALVEOLATA): A MULTI-GENE AND MULTI-GENOME APPROACH

(Contents of this chapter are in preparation for publication )

5.1. Abstract

Sequences from seven genes located on three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa. Nuclear sequences from the 18S rDNA, cytochrome oxidase c from the mitochondria; partial 16S and 23S rDNA sequences and two ribosomal polymerase sequences from plastid genomes were used. Maximum Parsimony, Maximum Likelihood and Bayesian

Inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in the nuclear gene phylogenetic trees. Analysis using the individual genes (18S rDNA and cytochrome c oxidase subunit I) reasonably resolved the various Apicomplexan groups with high

Bayesian posterior probabilities. The multi-gene, multi-genome analyses, based onl8S rDNA-Nuc, 16S-Plas, 23S-Plas, rPoB-Plas, rPoBl-Plas and COXI-Mit concatenated sequences, appeared to suggest that definitive host-parasite coevolution is relatively common within the phylum Apicomplexa, especially among eimeriid coccidia; coccidia infecting the digestive tract of a host probably evolved from parasites infecting the digestive tract of the ancestral host. The total evidence (consisting of a concatenation of all gene sequences) tree appeared to be useful in resolving phylogenetic relationships within the phylum Apicomplexa.

101 5.2. Introduction

The molecular phylogeny of the coccidia has been the subject of numerous studies

and opinions (Cavalier-Smith; 1983; Cavalier-Smith; 1993 Tenter et al, 2002). It has

however become necessary, in the light of recognized shortcomings of previous studies,

to explore more genes and genomes in order to have a better understanding of the

relationships within the phylum Apicomplexa, particularly with respect to the coccidia

(Tenter et al, 2002; Morrison et al., 2004; Morrison, 2008; Morrison, 2009).

The difficulty of establishing a widely accepted molecular phylogenetic

hypothesis for the phylum Apicomplexa is compounded by the fact that classical as well

as some current phylogenetic studies have been based on phenotypic characters or limited

molecular data (Siddall, 1995; Siddall et al., 1997). The molecular data for inferring

phylogeny in Apicomplexa have been limited in most cases to the nuclear genome and

frequently only 18S rDNA sequences (Carreno and Barta, 1999; Morrison et al., 2004;

Morrison, 2008). The difficulty of establishing positional homology among 18S rDNA

sequences across divergent taxa has complicated the use of these sequences for resolving phylogenetic relationships within the Apicomplexa and other phyla (Barta et al., 1997;

2001; Carranza et al., 1996; Li et al, 1997). Other nuclear genes that have been used to

investigate the evolutionary history of the Apicomplexa include 28S rDNA, ribosomal

ITS regions and adenylosuccinate lyase (Mugridge et al., 1999; Kedzierski et al, 2001;

Martinsen et al., 2008; and Samarasinghe et al., 2008).

Most members of the Apicomplexa possess functional nuclear, mitochondrial or mitochondria-derived and plastid genomes; however, some (e.g. gregarines) apparently lack a plastid genome and others (e.g. Cryptosporidium spp.) lack both plastid and

102 mitochondrial genomes (Wilson and Williamson 1997). Tenter et al. (2002) suggested that only analyses using multiple genes, preferably including both nuclear and organellar genes could generate a molecular phylogeny for the eimeriid coccidia that is likely to represent a reasonable organismal evolutionary hypothesis. The lack of comparable genes and genomes among some members of the Apicomplexa and variation in the evolutionary rates of different genes within genomes can be problematic. Molecular data generated from multiple genes and genomes should be subjected to parametric evolutionary models and robust phylogenetic analysis in order to establish homology in support of the interpretation of tree topologies as evolutionary histories, especially in the

Apicomplexa (see Morrison, 2008). The use of multiple genes and genomes for molecular phylogenetic and epidemiological studies in the Apicomplexa has been examined by some workers (Rathore et al., 2001; Perkins et al., 2007; Schwarz et al.

2009; Hikosaka, et al. 2010). Waller and McFadden (2005) examined the origin and function of the plastid in some members of the Apicomplexa. Phylogenetic studies involving members of the Apicomplexa have been carried out using plastid genes such as

ORF472 (open reading frame), CLPs (caseinolytic proteases), elongation factor Tu

(TufA) and ribosomal polymerases in the eimeriids and haemosporinids (Lang-Unash et al 1998; Blanchard and Hicks 1999; Cai et al 2003 and Saxena et al 2007). Zhao et al.

(2001) have suggested that the plastids might be a useful source of markers for the evolutionary delineation of apicomplexan taxa.

The commonest mitochondrial genes used for phylogenetic studies are cytochrome oxidases because of their universal occurrence in organisms that utilize oxidative phosphorylation as an energy source (Hebert et al., 2003; 2004a). There have

103 been few phylogenetic studies involving coccidia using mitochondrial or "mitochondria- like" genes (Schwarz et al, 2009) but mitochondrial gene sequences have been used extensively and successfully with haemosporinids and piroplasms (e.g. Putignani, 2004;

Omori et al., 2007; Perkins, 2008) for both molecular systematics and epidemiology.

Perkins et al. (2007) suggested that genes residing on each of the three genomes could be useful for constructing phylogenetic trees and can serve as a means of comparing evolutionary rates and patterns of sequence evolution through phylogenetic inference.

The use of total evidence (all available datasets) in the study of phylogeny is thought to be advantageous (Eernisse and Kluge, 1993; de Qiueroz et al. 1995). The purpose of this study was to evaluate genes from the three apicomplexan genomes (plastid, mitochondrial and nuclear) in the phylogenetic analysis of a number of coccidian parasites (Apicomplexa, Coccidia).

5.3. Materials and methods

5.3.1. Sources of Parasites and Parasite DNA

Oocysts of coccidia were obtained from a variety of sources. Laboratory strains of

Eimeria spp. from chickens (either single sporocyst or single oocyst derived lines maintained in our laboratory) were propagated in specific parasite free chickens in the

OMAFRA animal isolation facility (University of Guelph, Guelph ON, Canada).

Sporulated oocysts of laboratory strains of the murine parasites Eimeria falciformis and

E. papillata were kindly provided by Dr. Bill Chobotar, Andrews University, Berrien

Springs, MI, USA. Sporulated oocysts of the marsupial parasite, Eimeria trichosuri, were kindly provided by Dr. Michelle Power, Macquarie University, Sydney, Australia.

Sporulated oocysts of Cystoisospora suis from swine, Cystoisospora felis from domestic

104 cats and Eimeria zuernii from cattle were obtained from clinical fecal specimens submitted for diagnosis to the Animal Health Laboratory (University of Guelph, Guelph

ON, Canada). Sporulated oocysts were concentrated from fecal debris using standard salt flotation methods (Reid and Long, 1979) and finally stored in 2.5% potassium dichromate (w/v aqueous) at 4°C prior to DNA extraction.

Purified DNA from a laboratory culture of Toxoplasma gondii strain ME49 was kindly provided by Dr. J. P. Dubey, USDA, Beltsville, MD, USA.

5.3.2. DNA Extraction:

Sporozoites were excysted from sporulated oocysts by first grinding them to release sporocysts and sporozoites were released by incubation at 42 °C in excystation fluid containing either bile-trypsin for oocysts of Eimeria maxima or taurocholic acid-trypsin for other types of oocysts. DNA was extracted from cleaned sporozoites by using

DNAzol (GIBCO- Life Technologies, USA) according to the manufacturer's protocol.

DNA aliquots (140ng/ul) were prepared and frozen while working DNA was diluted to about 30ng/ul. PCR reactions were performed using 60-90 ng template DNA.

5.3.3. PCR

PCR was used to amplify specific plastid and mitochondrial genes using primers shown in Table 5.1.

105 TABLE 5.1. PLASTID AND MITOCHONDRIAL GENE PRIMER SETS

Product Genome Target Gene Primers Sequence Source (bp) Wang COI F GGTTCAGGTGTTGGTTGGAC ~810bp Wang COIR AATCCAATAACCGCACCAAG Cytochrome c COXI 10F GGWDSWGGWRYWGGWTGGAC This Mitochondria oxidase I ~500bp COX1 500R CATRTGRTGDGCCCAWAC Study (COI) COXI 10F GGWDSWGGWRYWGGWTGGAC ~780bp COXI 780R CATRTGRTGDGCCCAWAC Ribosomal RpoB2 F TCTAAGCCAAATGTATGGCAAG -728 polymerase B2 RpoB2 R CTTGAAGGAATACCTAAAGAATCAG Ribosomal RpoBlF ATTCTTTCACCTTGCCATAC -738 polymerase B1 RpoBl R GAACAAATAAATAGTTTTTCCG Cai et Plastid Ribosomal SSU F GCTCAGGAAAAACGCAAGAG al., 2003 -520 Small subunit SSU R TACATACGCTTTACGCCCAG Ribosomal LSU F AAATAGAAGTGAAAATGTCAGC -450 Large subunit LSU R TTTGATAAACAGTCGCTTGG

One unit of Platinum® Taq DNA Polymerase, 0.5mM dNTP's, 1*PCR buffer and

2.5 mM MgCb (Invitrogen, USA) was used to amplify ~90ng DNA template in each

50ul reaction. The reaction conditions were an initial denaturation at 95°C for 6 minutes

followed by 35 cycles of 95°C for 20 sec, 55°C for 30 sec and 72°C for lmin, with a final

extension at 72°C for 10 min. Both negative and positive template control reactions were

included with each PCR run. PCR products were electrophoresed on a 1.5% agarose

submarine gel in lxTris-acetate-EDTA (TAE) buffer at 120V for 45 min. The resulting

gel was stained with ethidium bromide and the size of products estimated by comparison

with a lOObp DNA size standard (Invitrogen).

5.3.4. Phylogenetic Analysis

5.3.4.1 Sequencing and Sequence Alignments

PCR products were either directly sequenced or purified from agarose gels.

Purification of PCR products was performed using Roche high pure purification kit

(Roche Applied Science, Germany) according to the manufacturer's instructions. PCR

cycle sequencing using the forward or reverse amplification primers followed by

106 detection on ABI Prism 3730 or 3100 DNA sequencers was completed by the Molecular

Biology Unit of the Laboratory Services Division, University of Guelph. Sequences for the 18SrDNA were obtained from GenBank. High quality contigs and final consensus sequences were generated using Geneious Bioinformatics software package (Version 5),

Biomatters Ltd., New Zealand (Drummond et al., 2010). Each cytochrome c oxidase subunit I sequence was approximately 500-810 base pairs in length depending on the primer set used to amplify this gene (Table 5.1). The 500 bp PCR products covered the

5" end of the 800 bp PCR product so all sequences were from the same region of the COI gene. The protein translation of the sequences presented open reading frames without stop codons. New (35) and existing published COI sequences were aligned using

Clustal-X (Larkin et al., 2007) and MAFFT (Katoh et al. 2009) implemented in the

Geneious (Ver. 5 and later versions) Bioinformatics software package (Drummond et al.,

2010). The 18S rDNA sequence alignment generated using Clustal-X and MAFFT was then edited manually by staggering the alignment and eliminating obvious misalignments according to the method described by Barta (1997). Sequences from multiple plastid genes from a single taxon were concatenated before single genome analyses.

Phylogenetic analysis of the 18S rDNA was performed using various apicomplexan taxa

(223 sequences representing 96 species) from various hosts. The individual sequences were: Toxoplasma gondii (U00458; EF472967; M97703) Neospora caninum (U03069;

AJ271354; AJ271354) Cystoisospora belli (U94787; AF106935); C. suis (U97523) C. ohioensis (AF029303); Isospora orlovi (AY365026); I. robini (AF080612; AF080612 );

Eimeria acervulina (EF210323; EF210324; EF175928; DQ538351; DQ136187;

FJ236372; U67115;) E. adenoides (AF324212) E. alabamensis (AF291427) E. albigulae

107 (AF307880) E. antrozoi (AF307876) E. arizonensis (AF307878) E. arnyi (AY613853)

E. aubumensis (AY876927); E. bovis (U77084) E. brunetti (U67116) E. catronensis

(AF324213) E. chaetodipi (AF339489) ; E. chobotari (AF324214) E. ahsata

(AF338350); E. faurei (AF345998); E. crandallis (AF336339); E. weybridgensis

(AY028972); E. ovinoidalis (AF345997); E. dipodomysis (AF339490); E. falciformis

(AF080614); E. furonis (AB329724); E. gruis (AB205165) E. langeberteli

(AF311640); E. leucopi (AF339491); E. meleagrimitis (AF041437) E. maxima

(EF210322; U67117; EU025110; FJ236332; FJ236330; FJ236331; EU025111;

FJ236334; EU025112; FJ236335; FJ263947; FJ236336; FJ236360; EF122251;

DQ538349; FJ236339; FJ236355; FJ236353; FJ236346; FJ236347; FJ236348 FJ236344;

FJ236354; FJ236345; FJ236358; FJ236359; FJ236338; FJ236343; FJ236350; FJ236352;

FJ236329; FJ236333; DQ538350; DQ640012; DQ136186 FJ236342; FJ236351;

EF210322; FJ236357; FJ236341; FJ236349; U67117; DQ538348; FJ236361; FJ236356;

FJ236340; FJ236337); E. mitis (EMU67118; U40262; U67118; FJ236379); E. mivati

(EMU76748; FJ236373; FJ236376; FJ236375; FJ236378; FJ236374; FJ236377); E. brunetti (U67116); E. praecox (FJ236362; FJ236367; FJ236366; FJ236368; FJ236370;

FJ236363; FJ236365; GQ421692; FJ236364; FJ236371; FJ236369; U67120); E. tenella

(U67121; EU025114; EF210325; DQ640011; EU025116; AF026388; DQ136180;

DQ136176; U40264; DQ136182; EU025113; DQ136184; DQ136179; DQ136177;

DQ136181; EU025115; DQ136178; DQ136183); E. necatrix (ENU67119; DQ136185);

E. separata (AF311643); E. nieschulzi (ENU40263); E. onychomysis (AF307879); E. papillata (AF311641); E. peromysci (AF339492); E_phalacrocoraxae (DQ398106); E. pilarens (AF324215); E. scabra (AF279668); Ejpolita (AF279667); E. porci;

108 (AF279666); E. reedi (AF311642); E. reichenowi (AB205177; AB205170); E. rioarribaensis (AF307877); E. scholtysecki (AF324216); E. sevilletensis (AF311644); E. telehi (AF246717); E. tropidura (AF324217); Caryospora bigenetica (AF060975;

AF060976); Choleoeimeria sp (AY043207); Cydospora cayetenensis (AFl 11183); C. cercopitheci (AFl 11184); C. colobi (AFl 11186); C. papionis (AFl 11187); Cydospora spp. NPL233 (U40261); Cydospora G22 (AF061566); Cydospora G34 (AF061567).

The mt COI sequences (93) include the following: E. acervulina (HM771673;

HM771674; EF158855; FJ236428; FJ236427; FJ236420; FJ236419; FJ236443); E. brunetti (HM771675); E. mitis (HM771681); E. mivati (FJ236433; FJ236441; FJ236434;

EF174185) E. maxima (FJ236432; FJ236440; HM771684; HM771685; HM771686;

FJ236410; FJ236436; FJ236414; EU025106; EU025105; EU025104; EF174184;

EF174183; EU025107; FJ236413; FJ236387; FJ236457; FJ236452; FJ236411;

FJ236459; FJ236454; FJ236450; FJ236439; FJ236429; FJ236408; FJ236394; FJ236380;

FJ236409; FJ236407; FJ236401; FJ236456; FJ236451; FJ236448; FJ236437; FJ236418;

FJ236412; FJ236402; FJ236417; FJ236390; FJ236406; FJ236435; FJ236393; FJ236389;

FJ236388; FJ236449; FJ236442; FJ236386; FJ236416; FJ236438; FJ236391; FJ236415;

FJ236392); E. necatrix (EU025108; HM771680) E. tenella (HM771676; HM771677;

HM771678; HM771679; EU025109; EF174188; EF174187; EF174186; FJ236404;

FJ236446; FJ236444; FJ236458; FJ236453; FJ236447; FJ236423; FJ236422; FJ236421;

FJ236430; FJ236424; FJ236400; FJ236396; FJ236395; FJ236384; FJ236381; FJ236445;

FJ236455; FJ236405; FJ236426; FJ236425; FJ236403; FJ236399; FJ236398; FJ236397;

FJ236385; FJ236383; FJ236382); E. falciformis (HM771682); E. vermiformis

(HM771683); E. zuernii (HM771687); N. caninum (HM771688); T. gondii (HM771689;

109 HM771690); (NC_009902); B. bigemina (AB499085); and

(AB499089).

The plastid gene sequences (35) consisting of up to four concatenated sequences of partial 16S rDNA and 23S rDNA sequences, and ribosomal polymerase (rpoB and rpoBl) sequences represented by the following taxa; T. gondii (GB ace No); T. gondii

(NC001799); Cystoisospora suis (GB ace No); E. trichosuri (GB ace No; E. acervulina

(GB ace No); E. falciformis (GB ace No); E. mitis (GB ace No); E tenella (GB ace No);

E. necatrix (GB ace No); E. maxima (GB ace No); E. brunetti (GB ace No); E. praecox(GB ace No); E. vermiformis (GB ace No); E. papillata (1: GB ace No; 2: GB ace

No); E. arizonensis (LSUAF307883); E. antrozoi (LSUAF307881); E. rioarribaensis

(LSUAF307882); E. sevilletensis (LSUAF332536); E. zuernii (GB ace No).

All new plastid sequences obtained were confirmed to be similar to known parasites of the same genus using nucleotide BLAST (Altschul et al. 1990) searches in GenBank.

Sequences were next assembled and aligned using Geneious (Drummond et al., 2010).

Alignment was initially performed using MAFFT and Clustal W (Katoh et al. 2009;

2005; Larkin et al. 2007) and later manually staggered and aligned by eye to address obviously spurious alignments and mismatches (Barta, 1997). Nucleotide alignments were translated to amino acids in protein-coding sequences to ensure that open reading frames existed without stop codons.

5.3.4.2. Data Analysis

A total of seven datasets were generated for the analyses. The first three datasets consisted of all publically available and newly generated sequences for Eimeria species and the outgroup taxa: Dataset 1) all available 18S rDNA sequences; Dataset 2) all mt

110 COI sequences; and Dataset 3) concatenation of up to four plastid genes (SSU, LSU, rpoB and rpoBl). The next three datasets were a subset of the first three datasets for only those taxa with a representative sequence (one or more) in all three genomes (i.e. the same species was found in all of the first three datasets). The final total evidence dataset from all genomes was constructed as follows; for each parasite species, a single strict consensus sequence was generated of its nu 18S rDNA, its mt COI and a concatenation of up to four of its plastid genes (described above). The resulting three consensus sequences for each genome for each parasite species (including ambiguity codes at variable positions among multiple sequences from a single genetic locus in a single parasite species) were concatenated into a single combined evidence dataset.

Analyses were performed using Maximum Likelihood and Maximum Parsimony in PAUP (Swofford, 2003) and Bayesian analysis with parameters estimated from model tests. Five million generations were run for the first three datasets (all 18S rDNA; COX1 and plastid sequences) whereas 1.5 million generations was sufficient for convergence in the remaining datasets. In all datasets analysed, a comprehensive model test was performed to determine the best nucleotide substitution rate model using MrModeltest

(Nylander 2004). In all tests, the hierarchical likelihood ratio test was employed and the best model selected using the Akaike information criterion.

5.4. Results

5.4.1.18S rDNA sequence analysis

The best-fit model for the global 18S rDNA dataset was the GTR+I+G. The ML,

MP and Bayesian analysis trees were similar but only the trees generated from Bayesian analysis and their posterior probabilities are shown. The MP tree had 223 taxa with a total

111 of 2023 characters weighted equally; 450 of these characters were parsimony informative. The tree length was 2430 with a consistency index of 0.5. The Bayesian analysis generated a consensus tree (see Fig. 5.1) in which members of the

Toxoplasmatinae (species of Cystoisospora, Neospora, Hammondia and Toxoplasma) formed a well supported monophyletic group that had a sister group relationship with coccidia of poikilothermic vertebrates (e.g. Hyaloklossia and Goussia spp.) and all other coccidia. Goussia metchnikovi and Eimeria tropidura found in fish and lizards respectively formed a well supported clade. Members of the Toxoplasmatinae and all of the early branching coccidia {Hyaloklossia and Goussia species plus Eimeria tropidura) illustrated in Fig. 5.1 possess valvular sutures on their sporocysts and do not have Steida bodies. Eimeria arnyi from a snake and E. ranae from frogs were early branching to a trichotomy comprised of i) Eimeria species of marsupials (E, trichosuri), ii) Eimeria spp. of cranes (E. gruis and E. reichenowi) and iii) the remaining coccidia. The genus

Eimeria was observed to be polyphyletic with at least four independent lineages of

Eimeria species being supported by the sequence data. Caryospora and Lankesterella species formed a monophyletic clade with 0.83 posterior probability support. Avian

Isospora and Atoxoplasma species formed a well supported monophyletic clade (1.00 posterior probability support - not shown in Fig. 5.1) although the monophyly of neither of these genera was supported. Eimeria species frequently formed well supported clades of parasites that parasitized the same or closely related definitive hosts such as Eimeria spp. infecting swine (1.00 posterior probability support), rabbits (1.00 posterior probability support) or galliform birds (1.00 posterior probability support) (see Fig. 5.1).

112 Eimeria alabamensis from cattle was the sister taxon to all Eimeria found in ruminants (mainly ovine) and lagomorphs and both were sister to species found in rodents or pigs. Cyclospora species formed a well supported monophyletic clade that was a sister group to a large clade of Eimeria species that infected piciform and galliform birds. Figure 5.2 represents the tree generated from Bayesian analysis of 18S rDNA sequences (95 sequences) from parasite species that also had both mitochondrial and plastid sequences. The MP tree length was 666 and the CI was 0.72 with 252 parsimony informative characters. All Eimeria spp. infecting domestic chickens formed a well supported monophyletic clade to the exclusion of other Eimeria species (E. trichosuri from marsupials and E. falciformis from mice). Monophyly of each Eimeria species infecting chickens was well supported with the exception of Eimeria tenella and Eimeria mivati.

113 0.2 Figure 5.1: Consensus Bayesian tree generated from 223 taxa of 18S rDNA sequences (GTR+I+G) with posterior probabilities of clade support. The same tree was obtained with MP analysis with a tree length of 2430 and a consistency index of 0.5. Enlarged views of boxed sections of the same tree are found on the pages indicated.

114 ' Hyalokloss'a lieberkhunu DH European green frog AF298623 Cystolsospo^a timoru DH Slendertailed MeerkatS suncattu EU2007S2 Cystoisospora tmom DH 5 suncatto AY279205 pCKSfOJSOSporasppDH feline {Gate) ABf?19675 ^~- Cystoisospora fehs DH Feline 176471 — Cystoisopora ohioensis DH Canine AY618555 - "•Cystottospornsms DH Porcine U97523 i "^Cystoisospora ohioensts DH Canine Al-029303 rt -Cystoisospora sp DH canine 1MMAB519674 •— Isosporaspecies DH TigerFJ357797 • Isospom oriovi DH Came! AY365026 • Hammondta hammondi DH Feline (Cats) AF096498 •Toxoplasma gondii DH Feline {Cats)U 12138 'loxop/asmc goncto DH Feline (Cats) L24381 ^Neospora camnumOH Canine () U03069 [mToxopiasma gondii DH Feline (Cats) U03070 fcloxop/crsmo good/f DH Feline (Cats) L37415 mHammondio heydomiGH Canine (Dogs) GQ984224 rHammondta heydomt DH Canine (Dogs) Baron_Cione3 T^Hammondta fteyrfom/DH Canine (Dogs) Baron_Clone5 FHommondfo tnjjittae DH Cantne (Dogs) GQ984223 *HammondiatnffittaeGH Canine (Cogs) GQ984222 Neospora cantnum DH Canine (Dogs)GQ899206 •Neospota cantnum DH Canine (Dogs) U 161S9 Neospora camnum DH Canine (Dogs) L24380 Neospora camnumOH Canine (Dogs) AJ271354 Neospora cantnum DH Bovine (Cattie) U17345 k Neospora cantnum DH Canine (Dogs) U17346

JOUASIC rnetchnikovi DH A'hstf finned gudgfon (Gobio of/jfp/nnoiys) FJ0CM234 ^Eimena tiopidura DH Hood Island Lizard [Tropidurus de/anon/s )AF3242177 Goussta neglectaCH Tadpoles of Rana nd.bunda and R escultrtaf}QG32$2 •Gowjiospa) Bufo bu/o_FJ009243 -Goussia noellen DH Frog tadpoles F<009241 s AB243084 Eimena (eich&nowi DH red-crowned crane, Gius japonensis A8?4^083 •Eimena reichenowi DH red-crowned crane, Grhs japonensis AB2Q5171 E

115 . o P ro^/sci i.i) A 3s > i;2 >i P^ A-30787c >ii Dh 3a ^ ATJO/S// nrto yc* ;i>s s DH Bib AFl Q~"9

•"< u i >< ft' r Lst '"F-nysci nap *A. J 3'T 1 L e a w;dp Di- i>pn orket ^rouse Ai jJMJ'9 rs s f>i R?*s ATJ07&'8 .*.* O-l Pas yiO/8%3 ffmtretimtro , utpvajm}$dipodjmyi s unDH K^JVHt f

LSI < DH U-s AT-* '215 B^te AJ32/P13 >s& Co >i ft/1 i r { r< tf}lc forma i>* Ife /m-iff tjs "i •& > ats A -*2 216 - "•/;*• / 0 ffir ir" w ^ I F f//re/ ire ek >t I em t<-c >ry — us At 2 0/ F rr-^njfoz * P J "e (• g^ i •/W rvnpn p,r r* l>i?< o6>* f)0H P^r tiMPi tt Etmena alabamensis DH CatHe F291427 fG/mrjOH Jv'ir'1 Sh Jr AF3 ra' ]f n" ow*>ido •• DH Sh p '"V * ••n sj AT *."S907 ff*DP oj/fs P*i Bo nt i r o ah^o a \>* Sfvtp (O-J •= C"e$ • 33d3 0 ybr iacns $ DH f f tp j" i iAji s DH c\ ^ iuj s i n era e%>dua >- aijjorp yi^ s Raji-j'Sj ^94009

f r.tr( eX/J! J DH 1 Tig jf! iJ s *\ibbt-v» i-Fo 4i / n'rap ' TS ">S Largo ^ pK Ciajjfc. rf69!01^

ncnu fjtftszi "S DH 1H 1 jjr ^ i ^abt ts 691011

9/ • DH L^i^ urpK'Mbuts FF6C4 JIS f rrira JP AV$* f T?or fhs RauN FKQ4010 Tpna pp fa an UH >rwrph Biub ,tKAi/ "iro J- (fUwLI 5-^940 6 00 merf TO< Lt.fj trfo Robhi-sirre"' tyc'ospara cayetarersi$ UH tthtopian monk^/s AF 11^*83 C>c?ospOfn spp DH Ethiopi?r inonk**y U4C261 Cycfo pora coiobi Dh Eth opian nw keys fifi111%> Cy<.'ospo'o <.ei.op tne^.1 DH EthiOp m r^onk«»jfa AF111185 C^/ospon ctropiii-L tlM Ethiopnnmof kc/s AFllllS^I Cy^loipoih papioms VH E hiopnn Monkeys ^F^.11187 Cvcic-pora spp DH Baboons G40 AT06i568 (yebspora spp [>H Bibcon^ G34 AhOSlSb/ (jciasiora spo 1MH Baboons O22A':0si5-)t> E/menosp DH Dendrocopos leucotos (white backed woodpecker) FN298443 Eimena tsnella DH Gallus domesticus (Domestic chicken) EF210325 -Eimena tenella DH Gallus domesbcus (Domestic chicken) DQ136183 - Eimena tenelta DH Gallus domesticus (Domestic chicken) DQ136181 • Eimena tenella DH Gallus domesbcus (Domestic chicken) DQ136180 " Eimena tenella DH Gallus domesticus (Domestic chicken) DCU36178 Eimena tenella DH Gallus domesticus (Domestic chicken) DQ136177 'Eimena tenella DH Gallus domesticus (Domestec chicken) DQ136179 Eimena tenella DH Gallus domesbcus (Domestic chicken) DQ136184 •Eimena tenella DH Gallus domesbcus (Domestic chicken) DQ136176 'Etmena tenella CM Gallus domesticus (Domestic chicken) DQ136182 •Eimena tenella DH Gallus domesbcus (Domestic chicken) EU025115 -Eimena tenella DH Gallus domesticus (Domestic chicken) EU025116 rEimena necatnx DH Gallus domesbcus (Domestic chicken) DQ136185 ^l— Eimena necatnx DH Gallus domesbcus (Domestic chicken) U 67119 Eimena tenella DH Gallus domesbcus (Domestic chicken) EU025113 ~Eimena tenella DH Gallus domesbcus (Domestic chicken) DQ.640011 Eimena tenella DH Gallus domesticus (Domesfcc chicken) EU025114 Eimena tenella DH Gallus domesbcus (Domestic chicken) U40264 Eimena tenella DH Gallus domesbcus (Domestic chicken) U67121

116 Eimena tenella OH Gallus domesbcus (Domestic chicken) EF210325 m£imena tenella DH Gallus domesbcus (Domestc chicken) DQ136183 "Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ136181 • Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ136180 Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ136178 Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ136177 'Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ.136179 'Eimena tenella DH Gallus domesbcus (Domestc chicken] DCH36184 'Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ.136176 'Eimena tenella DH Gallus domesticus (Domestc chicken) DQ.136182 Eimena tenella DH Gallus domesticus (Domestc chicken) EU025115 'Eimena tenella DH Gallus domesticus (Domestc chicken) EU025116 rFimena necabix DH Gallus domesbcus (Domestc chicken) DQ136185 ^""Eimena necabix DH Gallus domesbcus (Domestic chicken) U 67119 ^Eimena tenella DH Gallus domesticus (Domestc chicken) EU025113 Eimena tenella DH Gallus domesbcus (Domestc chicken) DQ640011 Eimena tenella DH Gallus domesbcus (Domestic chicken) EU025114 " Eimena tenella DH Gallus domesbcus (Domestc chicken) U40264 Eimena tenella DH Gallus domesticus (Domestc chicken) U67121 i '£m*f?7 Hl /lJ.7Cli f mt'-aaaen-'o oes DH TuHc^s AJFJ?^23/ e"osrp j» Mc e< g"i in opcjo HVJ11 /01- t m?> s L ti i/fcfayrs qaltrraso FM 701S pp DH V6?/p4,g/n ga Oi-i-o Hf/li/s, ? ! s " fp? o i % f Of v'« r gr s c,al pa JO n \ /D 4 •Winn OH f-Woys gallop H Jill Alia

pn UH P^o? ens-, eo to „5 HT 7008 T^rr ,o J1 ^as onus co'^ /s * 111 007 Eimena acervulina DH Gallus domesbcus (Domestc chicken) U571I5 Eimena acervulina DH Gallus domesbcus (Domestic chicken) EF210323 mena acervulina DH Gallus domesticus (Domestic chicken) EF17592S Eimena acervulina DH Gallus domesbcus (Domestic chicken) EF210324 Eimena acervulina DH Gallus domesbcus (Domestic chicken) FJ236372 Eimena acervulina DH Gallus domesbcus (Domestc chicken) DQX36187 Eimena acervulina DH Gallus domesbcus (Domestc chicken) DQ538351 Eimena mivab DH Gallus domesbcus (Domestic chicken) U76748 Eimena mivab DH Gallus domesbcus (Domestc chicken) FJ236378 Eimena mivab DH Gallus domesbcus (Domestc chicken) FJ236377 'Eimena mivab DH Gallus domesbcus (Domestc chicken) FJ236373 Eimena mivab DH Gallus domesbcus (Domestic chicken) FJ236375 'Eimena mivab DH Gallus domesticus (Domestic chicken) FJ236376 Eimena mivab DH Gallus domesbcus (Domestic chicken) FJ236374 Eimena mibs DH Gallus domesbcus (Domestic chicken) FJ236379 Eimena mibs DH Gallus domesbcus (Domestc chicken) U40262 Eimena mibs DH Gallus domesbcus (Domestc chicken) U67118 'Eimenapraecox DH Gallus domesbcus (Domestc chicken) FJ236366 ~Eimenapraecox DH Gallus domesbcus (Domestc chiclen) FJ236362 "Eimenapraecox DH Gallus domesbcus (Domestic chicken) FJ236363 Eimena praecox DH Gallus domestjcus (Domestic chiclen) U 67120 Eimena praecox DH Gallus domesticus (Domestic ch cten) FJ236365 ~Etmen'Eimenapraecox DH Gallus domesbcus (Domestic thicten) FJ236364 EEimena praecox DH Gallus domesbcus (Domestic chiclen) FJ236371 £" E" mena praecox DH Ga'Uis domesbcus (Domestic chiclen) FJ236370 Eimena praecox DH Gallus domesbcus (Domestic chiclen) FJ236369 L-t-£)f £,Eimei napraecox DH Gallus domesticus (Domestic chiclen) FJ236368 - t E mena praecox DH Gallus domesbcus (Domestic chiclen) FJ236367 099 r-Eimenaf praecox DH Gallus domesbcus (Domestc chiclen) GQ421692 • Eimena bametti DH Gallus domesbcus (Domestc chicken) U67116 ttn-e w maxima DH Gaijus dorresPcus (Domestic chicken} FJ23634? 'Eimena maxima DH Callus domesbcus (Domestic chicken; FJ236333 ™F mena maxima DH Gallus domesbcus {Domestic chicken) DQ1361% ™f mena maxima DH Gallus domesbcus iDumestit t hirken) H/36329 *h mena maxima PH Grllus domesticus (Domestic chicken! FJ236330 'i- mena maxima DH 6c us domesticus (Domestic chickenl FJ236331 rfimena maxima DH Gallus domesticus (Domestic chicken) FJ236332 fcr nena maxima DH Gallus domesbcus (Donvestic ch ckpn) FU02J110 I™ E mena maxima DH Gallus domesticus (Domestic chickeni DQ533350 I |~Eim'na maximt DH Gall is domesticus (Domestic chicken) EU025112 "1 ~~ Eimtna inax nw DH Gallus domesb us (Domestic chicken) FJ236335 I ••E/fmnofUQX/ua DH Gallus demesbcus (DomeitH. thicken) EU02511I *- I t/mP-"t na» maxEimena max ma DH Gallus domesbcus (Domestic chicken) FJ236334 EE met a maxima DH Gallus domesbcus (Domfstf chicken) FJ236336 P"^Eimena maxima DH Gal us domesbcus (Domestic chicken) fJ236361 I rEimenE mtna maxima DH Gallus doms'nus (Domestic chicken) FJ236338 1—IIV—f-

117 Limena acervutina DH Gallus domesticus (Domestc chicken) U67115 Eimena aceivulina DH Gallus domesticus {Domestic chicken) EF210323 , I F/me'JEm Q ocervu/mo DH Gollus domesticus (Domestc chicken) EF175928 If/Etmenan acervuhna DH Gal'us domesticus (Domestc chicken) EF210324 Eimena acervulina DH Gallus domesticus (Domestc chicken) FJ236372 Eimena acervulina DH Gallus domesticus (Domestic chicken) DQ.136187 Eimena acervulina DH Gallus domesticus (Domestc chicken) DQ538351 Eimena mivab DH Gallus domesticus (Domestc chicken) U76748 Eimena mivati DH Gallus domesticus (Domestc chicken) FJ236378 " Eimena mivati DH Gallus domesticus (Domestc chicken) FJ236377 ^Etmena mivati DH Gallus domesticus (Domestc chicken) FJ236373 Eimena mtvati DH Gallus domesticus (Domestc chicken) FJ236375 'Eimena mivab DH Gallus domesticus (Domestc chicken) FJ236376 " Eimena mivati DH Gallus domesticus (Domestc chicken) FJ236374 Eimena mitis DH Gallus domesticus (Domestc chicken) FJ236379 Eimena mitts DH Gallus domesticus (Domestc chicken) U40262 Eimena mttis DH Gallus domesticus (Domestc chicken) U 67118 Etmena pnjecox DH Gallus domesticus (Domestc chicien) FJ236366 mmEimena praecox DH Gallus domesticus (Domestc chicien) FJ236362 07- —Eimena praecox DH Gallus domesticus (Domestc chicten} FJ236363 'Eimena praecox DH Gallus domesticus (Domestic chicten) U67120 Eimena precox DH Gallus domesticus (Ctomestc thiclen) FJ236365 mmEim€Eimena praecox DH Gallus domesticus (Domestc chicien) TJ236364 ~Eimena praecox DH Gallus domesticus (Domestic chicten) FJ236371 Eimena praecox DH Gallus domesticus (Domestic chicien) FJ236370 Eimena praecox DH Ga'lus domesticus (Domestic chicten) FJ236369 Eimena praecox DH Gallus domesticus (Domestc chicien) FJ236368 Eimena praecox DH Gallus domesticus (Domestc chicien) FJ236367 099 tEimenaf praecox DH Gallus domesticus (Domestc chiclen)GQ421692 * Eimena brunew DH Gallus domesticus (Domestic chicken) U67116 '•imena maxima DH Gallus domesticus (Domestic < h cken) FJ236347 'Eimena maxima EM Ga"us aomesticus (Domestic chicken) FJ236333 'Eimena maxima OH Gallus domesticus (Domestic chicken) DQH»186 'Eimena maxima DH Ga'lus domesticus (Domestic chicken} FJ236"*29 Eimena maxima DH Gollus domesticus {Domestic chicken} FJ236330 Eimena maxina DH Gallus domesticus (Domestic chicken) FJ236331 r £ime"a rroxiva DH Go"us domes tit us {Domestc chicken) FM3£>332 ^Etmena maxima DH Callus domesticus (Domestc chicken) EU025I10 Eimena maxima DH Gallus domesticus [Domestic chicken) DQ.5383SQ Eimtna maxima DH Gallus domes tic ui (Domestc chicken) EU025112 ™ Eimena maxma DH Gallus domesticus (Domestc chttk^nj FJ235335 ™f mem; maxima DH Gallus domes ULUS (Domestc chicken) EU025I11 Eimena maxima DH Gal'us domesticus (Domestc chicken) FJ236334 E"rena maxima DH Gailus domesticus (Domestc chicken) FJ236336 Etmena maxima DH Galium domesticus (Domestic chicken) FJ236361 Etmena maxima DH Gallus domesticus (Domestc i.hickenj TJ236338 Eimena maxima DH Gallus domesticus (Domestic thicken) U67117 'tirreno maxima DH Gallus domesticus (Domestc f hie ken) EF21032? ^^ Eimena maxima DH Gallus domesticus (Domestc chicken) FJ236337 ~Eimena maxima DH Galium domesticus (Domestic chicken) FJ2363S8 p citnena maxima DH Gallus domesticus {Domestc chicken) F32363S9 ^~Eimena maxima DH Gallus aomesticus (Domestc chicken) PJ236341 _J^Cimena maxima Dti Gallus domesticus (Domestc chicken) FJ236339 J DWQ maxima DH Ganus domesticus (Domestc chicken? DQ040012 Lf£"Tie"0 max'ma DH Gai'us domesfcus (Domestc chicken) 00538349 ^ Enrena maxnra DH Gallus domesticus (Domestc chicken) EF122251 Eimena maxima DH Gallus domesticus (Domestic chicken) FJ23b3fe0 • Eimena max"ra IV Ga'lus doTesticus (Domestc chicken) 0Q538348 1 ^Eimena maxima DH Gallus domesticus (Domestic chicken) FJ236343 1—1 —Eimena maxima Df I Gallus domesticus (Domestc chicken) FJ236345 U r Eimena maxima DH Gallus domesticus (Domestc chicken) FJ236346 | "—Etmena maxima DH Gallus domesticus (Domestc chicken) FJ236344 '-••, Eimena maxima DH Gallus domesticus (Domestc chicken) FJ236357 'Eimena maxima DH Gallus domisticus (Domestic thicken) TJ23b3S2 mEtmena maxima DH Galium domesticus (Dames tn chicken) TJ236350 •Fimeaa maxima DH Galiu* domesticus (Dementi- chicken) FJ2363S3 •Eimena maxima DH Gallus domestic J\ {Domestc chicken) FJ73C3M kimena maxima DH Gollus domesticus (Domestr chicken) f i?36-J'>^ •— timena maxima DH Gallus domestinis (Domestic chicken) FI23634a "^—£fmena maxima DH Gallus domesticus (Domestc chicken) H236347 •— Eimena maxima DH Gallus domesticus (Domestc chicken) FJ236351 U— Eimena maxima Of I Gallus domesticus (Demesne chicken) FJ23fc340 0.2 ^Eimena maxima DH Gallus domesticus (Domestc chicken) FJ23&356 j~Etmena maxima DH Gallus domesticus (Domestic chicken) FJ236348 E,rreHo maxima DH Gailus domest><.us (Domestc chicken) FJ263947

118 - Toxoplasma gondii 1100458 Toxoplasma gondHM9rn(B Toxoplasma gondii EF472 967 • Neosp o ra caninum A J2 713 54 \tNeospora caninum U03069 0.8C| 1 CystoisosporafelisL76471 0-7^ Eimeria trichosun FJ829323 "^•Eimeria rtcftaswrf FJ829320 Eimeria falcifo mis AF080614 0.76 Eimeria tenella EF210325 •Eimeria ^ [Eimeria ' I •EimeriarEimei nitis U40262 0.98 Eimeria nitis U67118 Eimeria'Eimei brunettiV6'1116 r-Eimeria maxima FJ236329 •Eimeria maxima FJ236331 -Eimeria maxima FJ236330 0.94 •Eimeria mavimaF.1236332 -Eimeria maxima DQ136186 Eimeria maxima FJZ36333 1.00 rEimeria maxima DQ538350 'Eimeria maxima EU025110 I—Eimeria mav

Figure 5.2: Consensus Bayesian tree generated from 101 subset taxa (represented across all three genomes) of 18S rDNA sequences (GTR+I+G) with posterior probabilities of clade support. The same tree topography was obtained with MP analysis with a tree length of 666 and a consistency index of 0.71.

119 5.4.2. Cytochrome c oxidase subunit I sequence analysis

The GTR+G model was used for the analysis of COI sequences. Figure 5.3 shows the tree generated from the Bayesian analysis. Similar trees were generated using both

MP and ML analyses performed using 93 sequences made up of 833 characters of which

298 were constant, 115 were variable and 420 were parsimony informative; for the MP analysis, tree length was 1339 and CI was 0.61. The taxonomic outgroup were all members of the Toxoplasmatinae {Cystoisospora, Neospora and Toxoplasma spp.) and formed the sister group to the Eimeria species. Multiple sequences from a single Eimeria spp. all formed well supported monophyletic groups with markedly lower intraspecific genetic distances compared to the 18S rDNA tree described above with the exception of

E. mivati and E. mitis sequences that together formed a monophyletic clade but within which the monophyly of the individual species was not supported. Eimeria species from chickens did not form a monophyletic clade; Eimeria sp. sequences from other birds

(chukar, turkeys and pheasants) formed a weakly supported monophyletic clade that excluded the other Eimeria species of chickens. Eimeria acervulina formed a separate clade outside the E. maxima; E. necatrix; E. tenella and E. maxima clades. Figure 4 shows the tree generated by Bayesian analysis using 85 sequences and GTR+G as the best-fit model. MP analysis was performed using 833 characters, 311 of which were parsimony informative; the resulting tree had a length of 832 steps with a CI of 0.7.

Cystoisospora, Neospora and Toxoplasma species were used as outgroup taxa. As in the

18S rDNA tree, monophyletic clades of E. necatrix sequences and E. tenella sequences were sister clades.

120 1.00 I Neospom camnum NC1COX I T Toxoplasma gondii VHC10500 1.00 Toxoplasma gondii GTCO780 Cysloisopora felts mt COI ' Eimena sp ex Alecons graeca HM117020 Eimena zuemn COXWP ' Eimenasp MeleagnsgallopavoHM\170\8 - Eimenasp ex Phasianus cokhicus HM117017 Eimena sp Meleagns gallopavo HMU7019 1.00 r4 E'mena necatnx EU025108 Eimena necatnx GSWP Eime na tene lla MD1 Eimena lenella FJ236446 Eimena tenella FJ236444 Eimena lenella FJ236458 Eimena tenella FJ236453 Eimena tenella FJ236447 Eimena tenella FJ236423 Eimena tenella FJ236422 Eimena lenella FJ236421 Eimena tenella FJ236430 Eimena tenella FJ236424 Eimena tenella FJ236400 Eimena tenella FJ236396 Eimena lenella FI236395 Eimena lenella FJ236384 Eimena tenella FJ236381 Eimena lenella FJ236445 Eimena tenella FJ236455 Eimena tenella FJ23640S Eimena tenella FJ236426 Eimena tenella FJ23642S Eimena lenella FJ236403 Eimena tenella FJ236399 Eimena lenella FJ236398 Eimena tenella FJ236397 - Eimena lenella¥]236}$5 Eimena lenella FJ236383 Eimena tenella FJ236382 Eimena tnchosun COIGenBank l-00r Eimena falciformis COX WP Eimena vermlformis COXWP Eimena acervulma COXWP Eimena acervulma FJ236427 ' Eimena acervulma FJ236420 ' Eimena acervulma FJ236443 \Eimena acervulma FJ236428 Eimena acervulma FJ236419 Eimena brumIBGSCOXWP Eimena mivati FJ236441 Eimena mivati FJ236433 Eimena mivati FJ236434 Eimena mitis GSCOXWP Eimena /wva/rEF174185 0.60 Eimena maxima FJ236432 Eimena maxima FJ236440 Eimena maxima GS " Eimena maxima FJ236436 Eimena maxima EU025107 Eimena maxima FJ236413 1.00 Eimena maxima FJ236387 Eimena maxima FJ236459 Eimena maxima FJ236454 Eimena maxima FJ236450 Eimena maxima FJ236439 1.00 Eimena maxima FJ236408 Eimena maxima FJ236380 " Eimena maxima FJ236409 " Eimena maxima FJ236456 Eimena maxima FJ236451 Eimena maxima FJ236448 0.1 Eimena maxima FJ236437 Eimena maxima FJ236418 1" Eimena maxima FJ236412 °'yo " Eimena maxima FJ236390 " Eimena maxima FJ236435 Eimena maxima FJ236393 Eimena maxima FJ236388 Eimena maxima FJ236449 ~ Eimena maxima FJ236442 Eimena maxima FJ236386 Eimena maxima FJ236416 Eimena maxima FJ236391 [ Eimena maxima FJ236429 u Eimena maxima FJ236402 r Eimena maxima FJ236394 Eimena maxima FJ236417 f Eimena maxima FJ236407 Eimena maxima FJ236406 r Eimena maxima FJ236401 Eimena maxima FJ236389

Figure 5.3: Consensus Bayesian tree generated from 101 subset taxa cytochrome oxidase c I sequences (GTR+G) with posterior probabilities of ciade support. The same tree topography was obtained with MP analysis with a tree length of 1339 and a consistency index of 0.61.

121 Cystoisosporafehs mt COX1 l.OOr Neospora caramon NCI COX 1.00r ToxoplasmagondnFOiC\0500 r loxopiu .--0- - t Toxoplasma gondii GTCO7&0 0.98 - EimenaEimena falciformis trichasun COCOIGenBanX WP k 1 -Oflj Eimena necatnx EU025108 Eimena necatnx GSWP 0.91 Eimena tenella MD1 'Eimena tenella FJ2 36446 Eimena tenella FJ2 36444 ' Eimena tenella FJ236458 • Eimena tenella FJ236453 1.00 Eimena tenella FJ236447 Eimena tenella FJ236423 -Eimena tenella FJ236422 •Eimena tenella FJ236421 ' Eimena tenella FJ236430 Eimena tenella FJ236424 Eimena tenella FJ236400 " Eimena tenella FJ236396 'Eimena tenella FJ236395 •Eimena tenella FJ2 36384 0.59 0.92 ' Eimena lenellaF 12 36381 Eimena tenella FJ236445 Eimena tenella FJ236455 ' Eimena tenella FJ2 36405 Eimena tenella FJ236426 Eimena tenella FJ236425 'Eimena tenella FJ236403 Eimena tenella FJ236399 Eimena tenella FJ236398 Eimena tenella FJ236397 Eimena tenella FJ236385 1.00 Eimena tenella FJ236383 Eimena tenella FJ236382 Eimena acervuhna COXWP Eimena acervuhnaF123(A27 0.98 ' Eimena acervuhnaFJ2'S6420 0.1 " Eimena acervuhnaFJ23 64 43 [Eimena acervulinaF323642$ Eimena acervuhnaFJ236419 'Eimena bmnetti GSCOXWP Eimena mivati FJ236441 f~Eimena mivati FJ236433 0.95 f Eimena cf »wva((FJ236434 0.88 1.00U" Eimena mitis GSCOXWP Eimena mivati EF174185 Eimena maxima FJ236432 0.96 Eimena maxima FJ2 36440 Eimena maxima GS 'Eimena maxima FJ236436 ' Eimena maxima EU025107 " Eimena maxima FJ236413 1.00 ~ Eimena maxima FJ2363 87 ' Eimena maxima FJ236459 ' Eimena maxima FJ236454 ' Eimena maxima FJ236450 Eimena maxima FJ236439 1.00 'Eimena maximaFJ236408 ' Eimena maxima FJ2363 80 " Eimena maxima FJ236409 • Eimena maxima FJ236407 'Eimena maxima FJ236456 " Eimena maxima FJ236451 • Eimena maxima FJ236448 • Eimena maxima FJ236437 'Eimena maxima FJ236418 " Eimena maxima FJ236412 ' Eimena maxima FJ236390 - Eimena maxima FJ236406 " Eimena maxima FJ236435 ~ Eimena maxima FJ2 363 93 - Eimena maxima FJ2 363 88 - Eimena maxima F12 36449 ~Eimena maxima FJ236442 Eimena maxima FJ2363 86 ' Eimena maxima FJ236416 ' Eimena maxima FJ236391 [Eimena maxima FJ236429 Eimena maxima FJ236402 [Eimena maxima FJ236394 Eimena maxima FJ236417 r Eimena maxima FJ236401 Eimena maxima FJ2363 89

Figure 5.4: Consensus Bayesian tree generated from 85 subset taxa cytochrome oxidase c I sequences (GTR+G) with posterior probabilities of clade support. The same tree topography was obtained with MP analysis with a tree length of 833 and a consistency index of 0.7.

122 5.4.3. Plastid gene analysis

The best-fit model for the plastid gene dataset was the GTR+I+G. Figure 5.5 shows the tree generated from Bayesian analysis from 22 sequences. Both MP and ML gave the same trees. The MP analysis performed on the 22 taxa included 2502 characters, 1862 of which were constant, with a tree length of 1014 and CI of 0.73, and where 380 were parsimony informative. The sequences representing the four plastid genes, where available, were concatenated for each taxon. With members of the

Toxoplasmatinae as outgroup, the branching order of several of the Eimeria species used in the analysis was unexpected. Several Eimeria spp. from bats (E. arizonensis, E. antrozoi and E. rioarribaensis) formed a sister group to a number of Eimeria spp. found in mammals (rodents and herbivores) as well as, unexpectedly, Eimeria maxima. Eimeria brunetti was sister taxon to a well supported monophyletic clade consisting of E. necatrix and E. tenella. Figure 5.6 shows the Bayesian tree generated by analyzing the 13using the

GTR+I+G model. MP trees which were similar to the trees from Bayesian analyses included 2502 characters, a tree length of 831 and 314 parsimony informative characters.

The CI was 0.79. The tree shows E. maxima (chicken) coming up with E. falciformis

(murine) and the E. necatrix/tenella clade having a strong support.

123 Toxoplasma gondii cone, plastid sequences r ToxoplasmagondiiU87145 cone, plastid sequences "I 1.00 *•— Neospora caninum AF30431S cone, plastid sequences

Cystoisosporafelis cone plastid sequences 0.55 rt Cystoisospora suis cone, plastid sequences

~ Eimeria trichosuri cone, plastid sequences

Eimeria mitis cone, plastid sequences I 0.0.660 I— i 1.00 Eimeria acervulina cone plastid sequences

Eimeria brunetti cone, plastid sequences 0.89 "" Eimeria necatrix cone, plastid sequences l.oo \Eimt Eimeria tenella 80 cone, plastid sequences

Eimeria tenella AY217738conc plastid sequences

0.79 Eimeria arizonensis AF3325211 cone, plastid sequences 0.1 Eimeria antrozoi AF307881 cone, plastid sequences 0.92 L _. Eimeria rioarribaensis AF307882E cone, plastid sequences

0.83 0.88 - Eimeria zuernii plastid cone, plastid sequences I EimeriaEimi maximaconc plastid sequences

Eimeria papillata cone plastid sequences 0.80 — Eimeria nieschulzi AF332521 cone, plastid sequences

" Eimeria sevilltensis AF332536 cone, plastid sequences 1.00

0.70 I iEimeria falciformis cone, plastid sequences

1-001 EimeriaWi~ vermiformis cone, plastid sequences

Figure 5.5: Consensus Bayesian tree generated from 23 (GTR+I+G) concatenated available plastid sequences (LSU, SSU, rpoB1 and rpoB) with posterior probabilities of clade support. The same tree topography was obtained with MP analysis with a tree length of 1014 and a consistency index of 0.73.

124 Toxoplasma gondii 0.95

' Neospora caninum 1.00

^ Toxoplasma gondii RH

' Cystoisosporafelis

' Eimeria trichosuri

' Eimeria maxima

1.00 " Eimeria mitis

' Eimeria acervulina

0.88 " Eimeria falciformis

' Eimeria brunetti

1.00 ' Eimeria necatrix

1.00 Eimeria tenella

0.1 1.00 1 Eimeria tenella

Figure 5.6: Consensus Bayesian tree generated (GTR+I+G) from 14 subset taxa of concatenated plastid gene sequences (LSU, SSL), rpoB1 and rpoB) with their posterior probabilities. The same tree topography was obtained with MP analysis with a tree length of 831 and a consistency index of 0.79.

125 5.4.4. Multiple gene and genome consensus tree

Figure 5.7 shows the tree generated by Bayesian analysis of concatenated consensus sequences from all taxa that had at least one representative sequence in all genomes. From the 11 taxa that were used, the MP tree length was 1859 with 5124 characters of which 781 were parsimony informative with a CI of 0.76. The Eimeria species that infect birds formed a monophyletic clade that was sister to both E. trichosuri of marsupials and E. falciformis of rodents with which they formed a trichotomy.

126 1 Neospora caninum

1.00

Toxoplasma gondii

Cystoisosporafelis

Eimeria falciformis

Eimeria trichosuri

Eimeria acervulina

1.00

0.58 Eimeria mitis

0.1 0.58 Eimeria maxima

0.56 1.00 Eimeria brunetti

Eimeria necatrix

1.00

Eimeria tenella

Figure 5.7: Consensus Bayesian "total evidence" tree generated (GTR+I+G) from 11 taxa of concatenated strict consensus sequences from plastid (pis.) mitochondrial (mt.) and nuclear (nu.) gene sequences with their posterior probabilities. The same tree topography was obtained with MP analysis with a tree length of 1859 and a consistency index of 0.76.

127 5.5. Discussion

The use of single or multiple genes as markers in the molecular phylogeny of

organisms is an important aspect of molecular systematics (Avise, 1992). In this study,

we have generated novel sequences from the mitochondrial and plastid genomes of some

members of the Apicomplexa to evaluate the usefulness of a multigene and multigenome

approach to molecular phylogeny of the eimeriorinid apicomplexan parasites.

The use of multiple genomes and genes as genetic markers to study the molecular phylogeny of parasites has been the subject of many studies (Escalante and Ayala, 1994;

Siddall et al, 1997; Rathore et al., 2001; Kedzierski et al., 2002; Hagner et al 2007; Lau

et al., 2009; Bhoora et al, 2009; Hikosaka et al, 2010; Outlaws and Ricklefs, 2010).

Leander et al, (2003) suggested the further use of molecular data in the elucidation of the relationship between members of the Apicomplexa and their recent common ancestors.

An advantage of combining data from different genomes or different genes is that these

can be selected so that they differ in their rates of evolutionary change (Yang, 1996).

In this study, phylogenetic analyses using single gene sequences from nuclear

(18S rDNA), mitochondrial (COI) and concatenated plastid (SSU, LSU, RpoB and

RpoBl) genomes reflected some of the difficulties encountered when using genetic markers from single genomes such as difficulties in establishing positional homologies, particularly with the 18S rDNA sequences that are prone to large variations in sequence

length (see Barta, 2001). Analysis using a concatenation of consensus sequences from all

available genes in a total evidence approach appeared able to resolve clades of Eimeria

species in birds, marsupials and rodents more frequently than the use of sequences from

single genomes (see Gadagkar et al., 2005). Datasets corresponding to the three genomes

128 (nuclear, mitochondrial and plastid) were used individually and collectively to generate a molecular phylogeny of some apicomplexan parasites including the haemosporinids

(Perkins et al., 2007), some coccidia in the family Sarcocystidae (Votypka et al. 1998;

Slapeta et al. 2003 and Monteiro et al. 2007) and some Eimeria spp. (Zhao et al., 2001;

Zhao and Duszynzki, 2001).

In the present study, the trees generated from 18S rDNA sequences, both as a global alignment and as a subset of taxa represented across all three genomes, were largely able to discern individual species within the Eimeriidae using taxa in the

Sarcocystidae as taxonomic outgroups. However, the paraphyly of the genus Eimeria

(Tenter et al. 2002; Morrison et al. 2004) is evident in that clades of Eimeria spp. were interspersed with parasites belonging to other genera such as Lankesterella, Isospora,

Atoxoplasma and Cyclospora based on 18S rDNA sequences. The occurrence of

Atoxoplasma and Isospora spp. of avian origin in the same clade confirms previous studies (Barta and Carreno 1999; Barta et al. 2005; Schrenzel et al. 2005) suggesting that these coccidia of birds are closely related. The early branching of Eimeria arnyi and E. ranae, infecting colubrid snakes and frogs respectively, is in agreement with Jirku et al.

(2009). The placement of Goussia spp. and Eimeria tropidura (found in and reptiles) supports the hypothesis that suture-bearing (rather than Steida body bearing) sporocysts are a sympleisiomorphic character shared across many coccidial groups (see

Jirku et al., 2002) including the adeleorinid coccidia; in contrast, there is a single monophyletic clade of coccidia (most Eimeria spp., Cyclospora spp., Caryospora spp.) that all possess Stieda bodies in their sporocysts rather than sutures (Fig. 5.1) suggesting that this feature is a synapomorphic character for these parasites. Our large nu 18S rDNA

129 sequence based analysis corroborates several studies suggesting that the Stieda body of coccidian sporocysts is a reliable derived morphological trait that indicates membership in the eimeriid clade of parasites (Carreno and Barta 1999; Tenter et al., 2002; Jirku et al.,

2009).

The trees generated from both global and a subset of COI sequences formed monophyletic groups of members of the Eimeriidae and Sarcocystidae with members of the hemosporinids (Babesia and Theilerid) as outgroups and had obviously smaller intraspecific genetic distances and variation compared with the nuclear 18S rDNA sequence-based trees. This appears to be a useful feature of mitochondrial genes for both species delineation and phylogenetic resolution of the evolutionary history of the genome in the Apicomplexa as suggested by Ogedengbe et al. (2011) and Hikosaka et al. (2010).

The phylogenetic trees generated from concatenated plastid gene sequences representing all available taxa and as a subset (sequences with taxa across all three genomes) reflected in large part the grouping of parasites and their definitive hosts except for E. maxima of chickens which appeared in a clade with E. zuernii and E. papillata in the whole (all available plastid sequences) and with E. falciformis in a subset (sequences with taxa across the three genomes). In both trees, E. trichosuri isolated from a possum;

Trichosurus cunninghami (see Power et al., 2009), branched early from Eimeria from birds and mammals. The unstable position of E. maxima in the analyses was probably due to insufficient sequence data; it did not have the entire range of plastid sequences used for the other taxa.

A total evidence tree was generated from a concatenation of all available genes from the 18S rDNA, cytochrome c oxidase I gene and the four concatenated plastid

130 genes. Eimeria from marsupials (E. trichosuri) and rodents (E. falciformis) formed a

trichotomy with a monophyletic group that included all sampled Eimeria spp. from

chickens (c.f. Power et al., 2009). Even though we did not have representative taxa from

some of the Eimeria found in rodents, the observed branching order was similar to those

illustrated by Zhao and Duszynski, (2001) who primarily used the ORF470 plastid and

nuclear 18S rDNA in separate analyses for their studies. Zhao et al. (2001) showed that a

combined dataset including nuclear 18S rDNA and plastid 23S rDNA sequences could usefully delineate species that occur in rodents and bats.

From the foregoing, the most useful genomes for molecular phylogenetic studies

of Apicomplexa are the nuclear and mitochondrial genomes. Additional information

obtained from the plastid genome appears to be most useful as part of a concatenated

dataset involving all three genomes and the 6 genes sequenced from them. The

mitochondrial genes have the advantage of less intra-specific variations within taxa which has made them a potential barcoding gene (Ogedengbe et al., 2011). The partial plastid genes used in the present study, while useful, would probably present more reliable phylogenetic signal if the genes being used as markers were concatenated (see

Suchard et al., 2003; Gardagkar, et al. 2005). Both mitochondrial and plastid genes

appear to be maternally derived (Wilson and Williamson, 1997; Ferguson et al., 2005).

Stage-specific rRNA's of apicoplast and nuclear origin that are differentially expressed in various life cycle stages have been demonstrated for some apicomplexan parasites (see

McCutchan et al. 1995; Li et al., 1997; Ferguson et al., 2005). This results in paralogous sequences within some apicomplexan taxa (through gene duplication) that could confound phylogenetic analyses that are based on rDNA sequences.

131 The studies on the use of plastid or mitochondrial genes as markers for phylogeny in the Apicomplexa are few compared with that of the nuclear 18S rDNA gene. This study has contributed to the attempt at addressing this information gap by generating novel sequences from the three genomes to compare their usefulness for molecular phylogenetic studies and evaluate their use in a multigenome/multigene analysis. Most phylogenetic studies of the family Eimeriidae show a pattern of parasite-definitive host coevolution based mainly on nuclear genes (mainly 18S rDNA) but these studies suffer from problems associated with using a single gene as noted above and as suggested by

Martinsen et al. (2008). The observations in the present study indicate that a multigene approach could be useful in addressing both genomic and genetic evolution especially as it relates to parasite-host coevolution within and between members of the Apicomplexa that possess all three genomes, especially in addressing the polyphyly and paraphyly of economically important parasites such as those found in the Eimeriidae (See Hikosaka et al. 2010).

132 6.0. GENERAL DISCUSSION AND CONCLUSIONS

The phylum Apicomplexa is an important group of parasites whose identification,

classification and phylogeny have been the subject of numerous studies over the last

several decades. These studies have been limited in scope in terms of either the number

of taxa involved, the number of molecular markers employed and the limitation of

markers to mainly the nuclear genome (see Tenter et al., 2002). Further, some studies have been limited in terms of robust phylogenetic analyses involving model based

evolutionary changes within chosen markers. The eimeriid coccidia are important in

animal husbandry as well as for their zoonotic potential. One of the difficulties in the

study of members of the phylum Apicomplexa is accurate species identification and

characterization of isolates. Ghimire (2010), in a review of redescription of the genera within the family Eimeriidae, suggested that there are problems of taxonomy, reliable phenotypic characters, applicable phylogenetic characters, knowledge of life cycle patterns and demonstrated host specificities, that are exacerbated by many examples of pseudoparasitism. For example, the type species of the genus Isospora Schneider, 1881,

Isospora rara, was described as a pseudoparasite from a slug that had ingested avian feces. There is a recognized need for a more rational classification of important parasites of man and animals (see Ghimire, 2010). Putative identifications of the members of the

Apicomplexa were previously based on morphological characteristics, the location and appearance of lesions and type of hosts parasitized (Levine, 1988). Shirley (1975) discussed the unreliability of using morphological and pathological details in diagnosing oocysts because these features overlap considerably among Eimeria species. This recognition has driven the development of molecular methods for differentiating Eimeria

133 species that infect chickens (Morris and Gasser, 2006). Recently, these molecular methods have been used in an attempt to correlate flock performance with the epidemiology of Eimeria species (Schwarz et al, 2009). In this thesis, a multiplex PCR method developed by Fernandez et al. (2003a) was tested under field conditions in

Canada in order to assess its usefulness in confirming the purity of isolates for this study and its utility as an epidemiological tool for assessing prevalence of infections with

Eimeria spp. in local commercial poultry flocks. The SCAR markers which were developed from polymorphic regions of genomic DNA of isolated parasites were successful in Eimeria species identification in a limited epidemiological assessment of coccidiosis prevalence in farms in Ontario. Both single laboratory strains of Eimeria spp. maintained at OVC as well as mixed species field samples were successfully diagnosed using the method. The SCAR-based multiplex PCR could potentially be a useful tool for molecular epidemiology of coccidiosis in commercial flocks. The positive results obtained from samples of market-age chickens that were obviously fed with in-feed anticoccidials should encourage a more widespread and comprehensive sampling of flocks in Ontario for coccidiosis in order to evaluate both the status of the disease, vaccine efficacy and effectiveness of in-feed anticoccidials. This is important in the light of the dated information currently available regarding the prevalence of coccidiosis and the continued introduction of new forms of preventative methods that include vaccines that may or may not be effective in preventing coccidiosis in infected flocks. There is therefore a need to constantly monitor what species is(are) important in disease outbreaks or causes unthriftiness in infected flocks.

134 A good molecular epidemiological marker or gene for the purpose of species identification should be relatively conserved, heritable and a well characterized target

(e.g. ribosomal RNA/ITS regions or protein-coding genes for enzymes or structural proteins of known functions) that still varies enough for species differentiation rather than rely on random polymorphic regions of parasite DNA. Primarily because of uniparental inheritance and thus lack of complexity associated with recombination, the genomes of plastid and mitochondrion represent promising markers for this purpose. From experience with "non-" eukaryotes generally, and coccidia specifically, mt COI appears to be a useful genetic marker that is only now being exploited for identification of coccidial species (Hebert et al., 2004a; 2004b; Schwarz et al., 2009; Chapter 3).

DNA barcoding conceptually involves the use of a short (~700bp) DNA (usually the mitochondrion) region in order to specifically identify an organism at species level.

Barcoding is gaining recognition as a viable alternative to, or in conjunction with, morphological identification of organisms (Hebert et al. 2003). Mitochondrial COI is the most widely used gene for DNA barcoding of non-plant eukaryotes; this protein-coding gene within the mt genome is essential for oxidative phosphorylation. COI has been used for the discovery of potential new species in various parts of the world and is thought to be a useful gene for the eventual barcoding of all living things that possess this gene

(Hebert et al., 2003; 2004a). After comparing COI and the 18S rDNA sequences obtained during the research presented in this thesis, it became apparent based on genetic distances, species delimitation algorithms and species cluster trees using Bayesian and other phylogenetic analyses that COI is more effective in differentiating species than the

18S rDNA for coccidian parasites (Hulsenbeck and Ronquist, 2001; Ronquist and

135 Hulsenbeck, 2003; Tamura et al., 2007; Ross et al., 2008; Chapter 3). COI varied considerably less within species but varied more between species compared with the 18S rDNA sequences. This feature of the COI sequences indicates that the COI gene is a useful tool for diagnosing taxa, and perhaps identifying previously unrecognized taxa, in the phylum Apicomplexa broadly. The relative ease of establishing positional homology in the protein-coding COI sequences and the simple maternal inheritance of the mitochondrion makes the COI locus a convenient gene in molecular phylogenetics, either alone or preferably in conjunction with other genes. The utility of this locus will be greatly enhanced when sufficient taxa have been sequenced to overcome the likely saturation of the phylogenetic signal found within the third codon positions in the COI protein-coding sequences (see Blouin et al., 1998). Although nu 18S rDNA sequences will undoubtedly continue to provide phylogenetic information essential for resolving relationships among distantly related apicomplexan taxa, the COI locus may be more useful in resolving some of the phylogenetic problems inherent in using 18S rDNA sequences, such as for resolving the paraphyly of the coccidia in the genus Eimeria

(Chapter 3), The successful development of a single set of degenerate PCR primers in this thesis that can amplify mt COI from a wide range of apicomplexan parasites, including both eimeriid and isosporoid coccidia, has opened the possibility of DNA barcoding all coccidia and any coccidial life stages in tissue as well. This clearly needed taxonomic tool should prove to be of great utility for unraveling the uncertain taxonomic affinities of coccidia from a wide variety of vertebrate hosts and for linking tissues stages with enteric life cycles stages, including oocysts, for parasites for which the entire life cycle has not yet been elucidated. For example, the bloodstream forms of the adeleid

136 coccidia and the oocyst forms found within their (largely undiscovered) invertebrate

definitive hosts (see Siddall, 1995) could be connected through DNA barcoding of the

stages independently without the expensive (and frequently impractical) need to

demonstrate this relationship experimentally until molecular evidence of such a

relationship is generated. Even among relatively common coccidia of domesticated

animals, there are parasites such as for which the natural definitive

host is not known; screening oocysts from the feces of carnivores, omnivores and

scavengers using generic COI primers and sequencing may be a faster way of identifying

the natural definitive host for this parasite than biological assays. Also demonstrated in

this thesis was that the COI gene possessed sufficient phylogenetic signal to permit even

partial COI sequences to be used in molecular systematics, either alone or, preferably, in

combination with other loci. Combining data from a relatively quickly evolving locus

such as mt COI with a somewhat slower evolving 18S rDNA sequence, for example,

would provide phylogenetic signal that could support both deeper (older) evolutionary

relationships as well as species-level (younger) relationships. This combined dataset is

likely to be able together to resolve evolutionary relationships that neither dataset could

discover independently.

The 18S rDNA has been extensively used in the molecular phylogeny of members

of the Apicomplexa. The phylum Apicomplexa is thought to be monophyletic (Barta et

al, 1991) even though the placement of some members such as Cryptosporidium within the phylum remained uncertain (Barta, 1989) until recently when it has become generally

accepted that these parasites share a common ancestor with the gregarines (Barta and

Thompson, 2006; Templeton et al., 2010). The molecular phylogeny of parasites within

137 several less well studied apicomplexan groups continues to be problematic and amongst the least understood of these groups are the adeleorinid coccidia (Siddall, 1995). The problem of the adeleids is compounded by the fact that the systematics of this group has, until recently, been based almost entirely on the descriptions of blood stages of the parasite and this has led to a confused classification. Probably because of host sampling bias, most described adeleid parasites are haemogregarines belonging to any of six genera in three families with the most speciose genus being Hepatozoon. Hepatozoon spp. have vertebrate intermediate hosts and blood sucking such as acarines or insects as definitive hosts and vectors; to complicate matters, multiple intermediate hosts and transmission via predation are involved in the life cycle of some Hepatozoon spp., such as H. americanum (see Smith, 1996; Johnson et al., 2009). An examination of host- parasite coevolution amongst a variety of adeleid parasites was conducted in this thesis

(Chapter 4). Nuclear rDNA sequence data were subjected to detailed global and ingroup analyses of stagger-aligned sequences. These analyses provided evidence that there was strong association of monophyletic parasite clades with particular definitive hosts

(aquatic or terrestrial invertebrates) but this association did not exist for the vertebrate intermediate host (Barta and Desser, 1984; Vilcins et al., 2009; Chapter 4). Current understanding of some host-parasite relationships is that marine invertebrates were the earliest definitive hosts of apicomplexan parasites, but as life evolved to terrestrial forms there was need for the parasites to adapt to life outside the marine environment. Parasites that infected terrestrial invertebrates some of which then adapted to the new food source of vertebrate blood would then need to adapt to the new feeding preferences of the host

(vertebrate blood). In this way, invertebrate definitive hosts began transmitting the

138 parasites into intermediate hosts where the parasites undergo asexual development. This demonstrates for yet another group of apicomplexan parasites that definitive hosts are more important from an evolutionary perspective when compared to other types of hosts for the same parasites; the parasitizing of these non-definitive hosts is likely driven more by ecological, rather than coevolutionary, factors (e.g. Huff, 1938; Barta, 1989). Broader and deeper taxon sampling with complete 18S rDNA (possibly supplemented with sequences from other parts of the genome and from other genomes such as those from mitochondria and plastids) could help in confirming the monophyly of adeleid taxa.

More transmission experiments would also help to establish the definitive host for adeleorinid coccidia, particularly within the especially speciose genus Hepatozoon (see

Kimetal., 1998).

While single genes are highly useful as genetic markers in molecular phylogenetic studies, their phylogenetically informative characters may be best exploited by combining these data with data derived from other genes from the same taxa, preferably including genes that are under different selective pressures in a concatenated sequence analysis (Gadagkar et al., 2005). In research presented in this thesis, a combination of sequences from nuclear, mitochondrial and plastid genomes was shown to be useful in delineating the various groups within the phylum Apicomplexa. One challenge that may not be easy to overcome is the fact that mitochondrial and plastid genomes have not been demonstrated in some groups within the Apicomplexa. But wherever possible, more than one gene is probably necessary in addition to traditional biological identifications to study the phylogenetic relationships among species within the Apicomplexa (e.g.

Martinsen et al, 2008). For relative ease, 18S rDNA sequences would seem to be an

139 ideal gene for genus-level analyses in the Apicomplexa; this could be bolstered with ITS-

1 or ITS-2 sequences that have been shown to be of some use at the species and strain levels (Barta et al., 1998; Lew et al., 2003). Ancestral apicomplexan protists clearly predate many, if not all, metazoan eukaryotes and certainly all vertebrates that they now parasitize. This suggests that apicomplexan parasites likely exploited the newly arisen mammalian 'niche' as soon as mammals evolved. The subsequent divergence of mammals would have been a strong selective pressure on the protists infecting them

(with associated genetic changes) as mammalian populations evolved and speciated to occupy various ecological niches. In order to gain a more complete understanding of host-parasite coevolution and adaptation of parasites to their present ecological niches, as many genes as possible should be examined for their phylogenetic utility.

In this thesis, the utility of both random nuclear (Chapter 2) and well-characterized mitochondrial (Chapter 3) genetic targets for identification of coccidian parasites was explored. Although RAPD-SCAR generated markers were effective in examining the prevalence of Eimeria sp. infections in commercial poultry, a more generally applicable species identification marker is needed for broad use with a range of apicomplexan taxa.

Mitochondrial COI was shown to be such a marker with excellent utility for both species identification as well as molecular systematics. Multi-gene and multi-genome combined analyses using consensus-concatenated sequences from the three genomes (nuclear, mitochondrial and concatenated plastid) were shown to have higher clade support and resolution of monophyletic groups than single gene trees (Chapter 5) suggesting that future studies should sample multiple genes and genomes where feasible.

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171 8.0. APPENDICES

APPENDIX 1. COLLECTION AND INFECTION DATA FOR MARKET-AGE COMMERCIAL BROILER CHICKENS SAMPLED AT A COMMERCIAL POULTRY PROCESSING PLANT, 2007

Age of Fecal Farm Date Anticoccidial Usage Number of Eimeria species identified Birds Float ID Code Collected (+/-,Type»), Samples using Multiplex PCR (Days) Positive F APRIL 03 2007 32 Neo-Chlor/Oxy 100 10 0 0 N APRIL 03 2007 34 MxB 10 0 0 V APRIL 03 2007 35 3-Nitro 10 10 0 0 z APRIL 03 2007 34 MxB/MonB 10 0 0 2 E. tenella plus E. maxima; Af APRIL 17 2007 35 NA 10 3 1 E. maxima only Ag APRIL 18 2007 33 Diclazuril 10 0 0 B APRIL 18 2007 32 NA 10 0 0 Aa APRIL 18 2007 37 MxB/MoB 10 3 E. tenella Ab 35 NA 10 0 0 Ac MAY 01 2007 54 NA 10 0 0 Aj MAY 01 2007 23 NA 10 0 0 R MAY 01 2007 31 NA 10 0 0 X MAY 01 2007 37 NA 10 0 0 H 34 MxB 10 3 E. tenella and E. maxima D 45 NA 10 0 0 W MAY 02 2007 30 NA 10 3 2 E. tenella; 1 Eimeria maxima E MAY 02 2007 37 NA 10 0 0 P MAY 02 2007 ND MxB 10 0 0 U MAY 02 2007 30 NA 10 0 0 A MAY 05 2007 36 NA 10 0 0 Ad MARCH 28 2007 38 NA 10 1 E. acervulina Ae MARCH 28 2007 38 MxB 10 2 E. maxima Ah 40 10 3 E. tenella Ai MARCH 28 2007 35 MxB 10 0 0 C MARCH 28 2007 33 NA 10 0 0 G MARCH 28 2007 28 MxB 10 1 E. maxima I MARCH 28 2007 34 MxB 10 0 0 J MARCH 29 2007 ND BMO Maxi/Vinegar 10 0 0 K FEBRUARY 19 2007 32 MxB 10 0 0 L MARCH 29 2007 36 NA 10 0 0 M APRIL 19 2007 37 NA 10 0 0 0 APRIL 19 2007 32 MxB 10 0 0 Q APRIL 19 2007 42 MxB 10 0 0 s APRIL 19 2007 40 NA 10 0 0 T APRIL 19 2007 35 3-Nitro 10 0 0 Y APRIL 19 2007 31 MxB 10 0 0

'see Chapter 2 for description of anticoccidial products used in some flocks

172 APPENDIX 2: ALL NEWLY GENERATED SEQUENCES FROM APICOMPLEXAN PARASITES AND THEIR GENBANK ACCESSION NUMBERS

Genome, Gene and GenBank Accession Numbers Nuclear Mt Taxa Sequenced Plastid Genome (Species and Strain) Genome Genome SSU (18S) SSU (16S) LSU (23S) COI RPOB RPOB1 rDNA rDNA rDNA Klossia helicina Clone 2_6 HQ224955 ND ND ND ND ND Klossia helicina Clone 4_3 HQ224956 ND ND ND ND ND Babesiosoma stableri HQ224961 ND ND ND ND ND Dactylosoma ranarum clone 1A22 HQ224957 ND ND ND ND ND Dactylosoma ranarum clone IB 16 HQ224958 ND ND ND ND ND Haemogregarina balli HQ224959 ND ND ND ND ND Hepatozoon cf catesbianae HQ224954 ND ND ND ND ND Hepatozoon magna HQ224960 ND ND ND ND ND Hepatozoon cf clamatae B1 HQ224962 ND ND ND ND ND Hepatozoon cf clamatae B2 HQ224963 ND ND ND ND ND Neospora caninum strain NC-1 HM771688 Cystoisospora felis (######) Cystoisospora suis (#####) Toxoplasma gondii strain ME49 HM771690 (#####) (#####) Toxoplasma gondii strain GTI HM771689 Eimeria zuernii strain Guelph 2007 HM771687 (#####) (#####) (#####) (#####) Eimeria falciformis strain Chob2 HM771682. (#####) (#####) (#####) (#####) Eimeria papillata strain Chob (#####) (#####) Eimeria vermiformis strain Chob Eimeria mzY/sStrain USDA 50 ND HM771681 (#####) (#####) (#####) (#####) Eimeria mivati ND ND Eimeria acervulina strain NC3 ND HM771673 Eimeria acervulina strain USDA84 ND HM771674 (#####) (#####) (#####) (#####) Eimeria brunetti strain Guelph 80 ND HM771675 (#####) (#####) (#####) (#####) Eimeria maxima strain M6 ND HM771684 Eimeria maxima strain Guelph 74 ND HM771685 Eimeria maxima strain USDA 68 ND HM771686 Eimeria praecox ND ND Eimeria necatrix strain Guelph 84 ND HM771680 (#####) (#####) (#####) (#####) Eimeria tenella strain USDA 80 ND HM771676 (#####) (#####) (#####) (#####) Eimeria tenella strain MD1 ND HM771677 Eimeria tenella strain Guelph ID ND HM771678 Eimeria tenella strain Guelph 2D ND HM771679 Eimeria trichosuri ND (#####) (#####) (#####)

173