The Partial Mitochondrial Dna and Phylogenetic

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THE PARTIAL MITOCHONDRIAL DNA AND PHYLOGENETIC

RELATIONSHIPS OF THE ICELAND SCALLOP ( Chlamys islandica )

Victoria Northrup

Thesis

submitted in partial fulfillment of the

requirements for the Degree of

Bachelor of Science with

Honours in Biology

Acadia University

April, 2008

This thesis by Victoria Northrup

is accepted in its present form by the

Department of Biology

as satisfying the thesis requirements for the degree of

Bachelor of Science with Honours

Approved by the Thesis Supervisor/ Head of the Department

______Dr. Marlene Snyder Date

Approved by the Honours Committee

______Dr. Anna Redden Date

______Dr. Glenys Gibson Date

______Dr. Liza Duizer Date iii

I, Victoria Northrup, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I however, retain the copyright in my thesis.

______Signature of Author

______Date

Acknowledgments

I would like to thank Duncan Bates and Dr. Mike Dadswell for supplying the scallops used in this study. Thank you to the Howard Gould Trust microbial genome sequencing initiative for funding this research. I would like to acknowledge Dr. Marlene Snyder for her input on this study.

Table of Contents Page Title page ...... i Approval page ...... ii Permission for duplication page...... iii Acknowledgements ...... iv Table of contents ...... v List of Tables ...... vii List of Figures ...... viii Abstract ...... x Chapter 1: Introduction ...... 1 1.1 Chlamys islandica life history and taxonomy ...... 1 1.2 Mitochondria ...... 5 1.3 Mitochondrial Genome ...... 8 1.4 Secondary Structure and Compensatory Mutation ...... 10 1.5 Purpose of study ...... 13 Chapter 2: Sequence Analysis ...... 13 1 Introduction ...... 13 2 Material and Methods ...... 15 2.1 Sample collection ...... 15 2.2 DNA isolation ...... 15 2.3 Gene amplification ...... 16 2.3.1 Primers ...... 16 2.3.2 Polymerase chain reaction ...... 17 2.3.3 Gel electrophoresis, band visualization and excision ...... 19 2.3.4 Isolation of DNA from agarose gel ...... 19 2.3.5 DNA sequencing ...... 20 2.3.6 Sequence comparison ...... 21 2.3.7 Amino acid sequence ...... 21 2.4 Whole genome amplification ...... 21 2.4.1 Primer design ...... 22 2.4.2 Long accurate polymerase chain reaction ...... 22 2.4.3 Gel electrophoresis, band visualization and excision ...... 23 2.4.4 Isolation of DNA from agarose gel ...... 23 2.4.5 DNA sequencing ...... 23 2.4.6 Sequence comparison ...... 23 3 Results and Discussion ...... 24 3.1 Gene amplification ...... 24 3.2 Amplification of long accurate PCR fragments ...... 42 Chapter 3: Phylogenetic Analysis ...... 46 1 Introduction ...... 46 2 Material and Methods ...... 49 2.1 Sequence analysis ...... 49 2.2 Phylogenies ...... 50 2.2.1 Obtaining the sequences ...... 50 2.2.2 Creating phylogenies ...... 50 vi

3 Results and Discussion ...... 55 Chapter 4: Conclusions...... 73 Chapter 5: References ...... 75 Appendix 1 ...... 82 Appendix 2 ...... 83

vii

List of Tables Page Table 1. Primers used in the amplification of mitochondrial genes for PCR ...... 17 Table 2. The optimal PCR conditions for each gene ...... 18 Table 3. PCR cycling parameters for each gene ...... 19 Table 4. Primers used in successful LAPCR ...... 22 Table 5. The GenBank accession numbers and references for the 39 scallops and the outgroup of donkey’s foot ( Spondylus gaederopus ) ...... 51 Table 6. The GenBank accession numbers, classification and references for 13 bivalves with complete mitochondrial genomes sequenced and a Polyplacophora that is used as an outgroup for the phylogenies ...... 55 Appendix 1 Table 7. All primers used in the study, their target and primer sequence ...... 82 Appendix 2 Table 8. The GenBank accession numbers for genes sequenced in this study ...... 83

viii

List of Figures Page Figure 1. 16S amplification ...... 25 Figure 2. CytB and 12S amplifications ...... 26 Figure 3. COIb amplification ...... 27 Figure 4. COIa amplification ...... 28 Figure 5. Nad1 amplification ...... 29 Figure 6. Nad3 amplification ...... 30 Figure 7. Nad4 amplification ...... 31 Figure 8. Atp6 amplification ...... 32 Figure 9. The edited partial sequence for the 16S (lrRNA) of 196 nucleotides ...... 32 Figure 10. The edited partial sequence for the 12S (srRNA) of 412 nucleotides ...... 33 Figure 11. The edited partial sequence of COIb of 234 nucleotides ...... 34 Figure 12. The edited partial sequence of COIa of 242 nucleotides ...... 34 Figure 13. The edited partial sequence of the CytB gene of 287 nucleotides ...... 35 Figure 14. The edited partial sequence of the Nad1 gene of 223 nucleotides ...... 35 Figure 15. The partial edited sequence of the Nad3 gene of 167 nucleotides ...... 36 Figure 16. The edited partial sequence of the Nad4 gene of 340 nucleotides ...... 36 Figure 17. The edited partial sequence of the Atp6 gene of 286 nucleotides ...... 37 Figure 18. The hypothetical amino acid sequence for COIb gene ...... 38 Figure 19. The hypothetical amino acid sequence for COIa gene ...... 39 Figure 20. The hypothetical amino acid sequence for CytB gene ...... 39 Figure 21. The hypothetical amino acid sequence for Nad1 gene ...... 39 Figure 22. The hypothetical amino acid sequence for Nad3 gene ...... 40 Figure 23. The hypothetical amino acid sequence for Nad4 gene ...... 41 Figure 24. The hypothetical amino acid sequence for Atp6 gene ...... 41 Figure 25. 12S-Nad1 amplification ...... 43 Figure 26. The edited sequence for the fragment 12S-Nad1 of 352 nucleotides . . . . . 44 Figure 27. Putative secondary structure for the tRNA for serine ...... 45 Figure 28. Phylogenetic relationship of 39 scallop species and Spondylus gaederopus as an outgroup using the partial 16S rRNA sequence ...... 60 Figure 29. Phylogenetic relationship of 30 scallop species and Spondylus gaederopus as an outgroup using the partial 12S rRNA sequence ...... 61 Figure 30. Phylogenetic relationship of 39 scallop species and Spondylus gaederopus as an outgroup using the partial 12S rRNA and 16S rRNA sequences ...... 62 Figure 31. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata , using the partial sequence of the COI gene (b) ...... 65 Figure 32. Phylogenetic relationship of 13 bivalves with no outgroup using the partial sequence of the COI gene (a) suspected of being a pseudogene . . . . 66 Figure 33. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad1 gene ...... 68 Figure 34. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Cytb gene ...... 69 Figure 35. Phylogenetic relationships of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad3 gene ...... 70 ix

Figure 36. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad4 gene ...... 71 Figure 37. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the ATP6 gene sequence ...... 72

Abstract

The taxonomy of the family Pectinidae has been called in question due to recent molecular evidence coming to light. The mitochondrial genomes of many molluscs are unusual and defy many of the conventions thought normal of animal mtDNA. The genes found in the mitochondria, including the LSU and SSU rRNA genes, are readily used for deriving potential phylogenetic relationships. Individual gene amplification was done using PCR and whole genome amplification was attempted using long accurate PCR.

The gene order of 12S-Nad1 was found, possibly with trnS in between these two genes.

The sequences obtained were used to create phylogenies of the LSU and SSU rRNA for the family Pectinidae and protein coding genes (COI, CytB, Nad1, Nad3, Nad4, and

Atp6) were used to create phylogenies for the class Bivalvia. Chlamys islandica is found to be closely related to Mizuhopecten yessenosis in the Pectinidae phylogenies. The genus of Chlamys, like most other genera in the family Pectinidae, is not monophyletic but is polyphyletic. In most of the bivalve phylogenies Chlamys islandica also groups with Mizuhopecten yessenosis and the family Pectinidae is monophyletic. The phylogeny based on Atp6 gives anomalous results compared to the other phylogenies. The pseudogene for the COI also does not agree but the actual gene does agree with the monophyly of Pectinidae and the close relatedness of Mizuhopecten yessenosis and

Chlamys islandica.

Chapter 1. Introduction

1.1 Chlamys islandica life history and taxonomy

Chlamys islandica , more commonly known as the Iceland scallop, is a subartic species and can be found around the coast of Norway, Iceland, Greenland and eastern

Canada (Ekman 1953). They are characterized by their chlamyoid shape which is defined by: the height of the shell exceeds the length, and the anterior outer ligament exceeds the posterior outer ligament. Some of their morphological characteristics are considered primitive, such as their ribbing which is simple crenulation, known as pilace, without internal carinae. These resemble simple waves (Shumway 1991).

Chlamys islandica is the northernmost species in the family Pectinidae. They are found in the northern boreal waters (Ekman 1953), from depths as shallow as 10 m to as deep as 250 m and tend to be more concentrated in water under 100 m. They live most commonly in coarse sediments that consist of sand, gravel, shells and on occasion clay

(Shumway 1991).

Chlamys islandica is a dioescious species and when ripe the ovaries are pinkish in color and the testis are white in color. The gonads do vary in size seasonally with the most rapid weight gain of the gonads of both sexes from March to May. The testis are heavier than the ovaries. The majority of scallops become sexually mature within 3-6 years of age where the fastest growing become sexually mature earlier than the slower growing. Spermatogenesis and oogenesis occur from March to October (Shumway

1991). The larger scallops also tend to spawn first, followed by the smaller scallops. The spawning process is triggered by short term variations in temperature which are brought 2 about by vernal melt water discharge. The gonads are fast to recover. Chlamys islandica exhibit external fertilization and are very fertile (Shumway 1991).

Chlamys islandica produce planktotrophic larvae that usually spend around 10 weeks in the plankton from June to August as such before settling. Like other scallops, they also have a trochophore larval stage, termed a prodissoconch, that consists of two stages, prodissoconch I and prodissoconch II. During this stage the shell begins to form

(Shumway 1991). During the prodissoconch I phase, a stellate microsculptural pattern without distinct comarginal growth lines is apparent on the external surface. The hinge teeth have not yet developed at this stage; these develop in the prodissoconch II. The comarginal lines also occur in prodissoconch II (Shumway 1991).

The dissoconch, also known as the metamorphic line (Carriker and Palmer 1979), separates the larval (prodissoconch) and juvenile parts of the shell. The dissoconch is characterized by a sharp line of demarcation from the succeeding post-larval stage

(Shumway 1991). During metamorphosis the velum is lost and there is rapid growth in the size of the foot, the posterior adductor and the gills. There is also an elaboration of the innermost mantle fold and its sensory structures. The shell morphology also changes with the first increments of the dissoconch that reveal the beginning of the byssal notch of the right valve as well as the auricles and disks of both valves (Shumway 1991).

Once metamorphosis is complete the scallop is ready to continue development of the monomyarian condition and the pleurothetic mode of life with the right valve against the substrate or attachment surface (Shumway 1991). The microstructure and mineralogy of the shell also changes. The outer shell develops foliated calcite on the left valve

(Shumway 1991) which gives rigidity (Waller 1972). The right valve forms columnar 3 prismatic calcite (Shumway 1991) which gives flexibility (Waller 1972). The scallop has now entered the prismatic stage. The flexibility in the right valve allows the margins of the right valve to flex against the rigid left margins. In the Chlamys genus this stage is usually 2 µm (Shumway 1991).

In the next stage, the pre-radial stage, the prismatic calcite on the right valve disappears along with the beginning of the radial ribs since they interlock at the margin.

The hinge teeth, muscle scars and growth surfaces of the microstructural layers of the shell can be found on the inner surface of the shell and are important morphological features used in taxonomy and phylogeny of scallops (Shumway 1991).

Chlamys islandica shows seasonal growth rates with the spring showing the highest growth rates due to the plankton bloom, supporting a higher metabolic rate

(Shumway 1991). Growth rates also vary by location of the population, with the population in Balsford, Norway being reported to grow 10-11 mm/year in shell height in the first six years of life (Brun 1971). This population shows the fastest growth rate exhibited by Chlamys islandica . They can grow from 39 mm at 5 years of age to 82 mm by 10 years (Wigborg 1962; Wigborg 1963). In the northwestern Iceland population they can grow from 50 mm to 84 mm from 5 to 10 years of age (Eiriksson 1988). Populations in other areas of Norway can exhibit growth from 30-35 mm to 70 mm from 5 to 10 years of age (Vahl 1981; Wigborg 1962; Wigborg 1963) while Chlamys islandica in northern

Spitsbergen populations can grow from 30 mm to 60-65 mm and Chlamys islandica from

Mitra Bank can go from 25 mm to 50 mm over the same age span (Wigborg et al . 1974).

The northern populations usually exhibit more overall growth than the southern populations. The age of the scallop also affects the growth rate with greater amounts of 4 growth occurring during the first six years. Population densities also vary by location.

The Bear Island population averages 10 scallops/m 2 and other populations exhibit densities of upwards of 60 scallops/m 2 (Shumway 1991).

Age distribution of the populations also varies by location with those found in

Norway varying from 2-12 years with the majority of the population being eight years or older (Wigborg 1963). In western Greenland 40% of the population have been reported as being over 21 years of age (Shumway 1991). The age of the scallops can be measured by two methods. Wigborg (1963) obtained age by counting the number of dark and light bands on the shell surface. Johannessen (1973) found it more accurate to measure the lines produced on the ligament (resilium). Populations of Chlamys islandica tend to be made up of more large scallops than small with the average being over 60mm, but sizes range from 10-100mm (Shumway 1991). This could be due to predation because size is related to vulnerability (Arsenault and Himmelman 1996).

Chlamys islandica , like other scallops, are filter feeders. They are capable of swimming short distances, with the medium-sized scallops being the better swimmers

(Shumway 1991). The enhanced swimming could be due to predator avoidance behavior as scallops in this size range (from 15-30mm) are more commonly preyed upon. Those above 60mm are rarely preyed upon (Arsenault and Himmelman 1996). Their natural predators include the sea stars ( Asterias rubens ), cod and eider ducks ( Somateria spp. )

(Shumway 1991) and crabs ( Hyas araneus and Cancer irroratus ) (Arsenault and

Himmelman 1996).

Chlamys islandica belongs to the domain Eukarya, kingdom Animalia, phylum

Mollusca, class Bivalvia, subclass Pteriomorphia, order Pterioida, family Pectinidae, 5 genus and species Chlamys islandica . There are three subspecies: Chlamys islandica costellatus, Chlamys islandica inscultus and Chlamys islandica islandica (ITIS Report

2007). Scallops are believed to have evolved from the family Entoliidae (Teppner 1922).

The family Entoliidae are non-ctenoliate Pectinacea that evolved during the mid-

Paleozoic and flourished in the waters during the Mesozoic and may have become extinct at the Cretaceous-Tertiary boundary due to a meteorite strike about 65 million years ago

(Shumway 1991). The first Pectinidae were ctenolium-bearing scallops that emerged in the Triassic period during the taxonomic reconstruction of marine biota that followed the extinction at the end of the Paleozoic era 230 million years ago. Two groups emerged, one with radial ribs or riblets on both disk and auricles and the other with smooth shells

(Shumway 1991) which belonged to the genus Pleuronectities (von Scholothiem 1823) that are believed to be the ancestors of the Camptonectes (Meek 1864). Camptonectes was a genus that developed prominent microsculpture but did not have well-developed ribbing and belonged to the Jurassic and Cretaceous period (Johnson 1984).

Praechlamys was the first genus to have prominent ribbing (Allasinaz 1972).

They were the first Chlamys -like taxa to evolve in the Mesozoic Era (Shumway 1991).

In the late Triassic the chlamyoid appearance had occurred characterized by a pattern of marginally interlocking ribs that were generally branched on the right valve and intercalated on the left, similar to the modern Chlamys genus (Shumway 1991).

1.2 Mitochondria

Mitochondria are semi-autonomous organelles found in eukaryotic cells that act as a “power-house”(Andersson 2003; Scheffler 2001; Scheffler 1999). They have their own genome that encodes some of the proteins used in the mitochondria as well as the 6 transcription and translation machinery. They produce adenine triphosphate (ATP) through oxidative phosporylation using the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle or citric acid cycle) and the electron transport chain that allows for respiration (oxygen is the terminal electron acceptor in aerobic organisms) (Boore 1999;

Karp 1999; Scheffler 2001; Scheffler 1999). Glycolosis occurs in the cytoplasm of the cell and the pyurvate is imported to the mitochondria where it is decarboxylated to form acetyl CoA (Karp 1999; Voet et al . 2006). The acetyl CoA enters the TCA cycle which is located in the matrix of the mitochondria with the exception of succinate dehydrogenase which is found in the inner membrane. This produces FADH 2 and three

+ NADH and GTP (as well as HS-CoA and 3 H ). The NADH, FADH 2 and GTP will then continue to the electron transport chain (ETC) that is embedded in the inner membrane

(Voet et al . 2006).

The number, shape and size of mitochondria per cell can vary depending on the type of cell, from a few hundred mitochondria to a few thousand. The mitochondria have two membranes, the outer membrane and the inner membrane. The outer membrane contains a 50:50 ratio of proteins to lipids by weight which is typical of eukaryotic membranes, but the inner membrane has a ratio of 75:25 proteins to lipids which is typical of the bacterial cell membrane (Karp 1999; Scheffler 2001; Scheffler 1999). The inner membrane is divided into two domains (but is a continuous membrane). Part of it is close to the outer membrane; this part is called the inner membrane boundary. The inner membrane also folds inward to form cristae. Cristae are tubular or lamellar foldings that are continuously connected to the inner boundary membrane by small tubular structures called cristae junctions. This division also gives rise to two compartments of the 7 mitochondria, the intermembrane space and the intracristae. The mitochondrial matrix is located in the intracristae which is the space between the cristae (the inside of the mitochondrion) (Scheffler 1999). The intermembrane space is the space between the inner and outer membrane. There tends to be more cristae (i.e. greater surface area) in muscle cells and neurons, which are the cells that require greater amounts of respiration and therefore more energy (i.e. ATP) (Scheffler 2001; Scheffler 1999).

It is widely accepted that mitochondria arose through an endosymbiotic relationship (Andersson 2003; Ballard et al . 2004; Boore 1999; Boore and Brown 1998;

Emelyanov 2001; Gray et al . 2001; Karp 1999; Lang et al . 1999; Scheffler 2001;

Scheffler 1999; Yang et al . 1985). The endosymbiont theory states that a free-living bacterium entered into an endosymbiotic relationship (the ancient eukaryotic cell is not well characterized). The free-living bacteria has been determined to be an α- proteobacteria from the order Rickettsiales (Andersson 2003; Emelyanov 2001; Yang et al . 1985) which include the family Rickettsiaceae and several Rickettsia -like endosymbionists (RLEs). Rickettsiae are a group of the α subdivision of proteobactia and are obligate intercellular endosymbionts of eukaryotic cells. The monophyletic relationship between mitochondria and the order Rickettsiales has been shown in many independent phylogenies based on conserved proteins and ribosomal ribonucleic acid

(rRNA) (Emelyanov 2001).

The endosymbiotic relationship is speculated to have occurred two billion years ago when atmospheric oxygen levels were rising. At this time aerobic bacteria

(cyanobacteria) were photosynthetically active, causing the levels of oxygen to rise due to photosynthesis. Before this time, the atmosphere of earth would not have been oxygen 8 rich, but would have contained low levels of oxygen and consisted of carbon dioxide along with reduced forms of sulfur, nitrogen and carbon. The oxygen increase could have caused a major crisis on earth. Anaerobic life forms that were unable to protect themselves from the oxygen or find a microenvironment devoid of oxygen would have gone extinct. The higher levels of oxygen resulted in early eukaryotes forming an endosymbiotic relationship with the α-proteobactia that could use oxygen as the terminal electron acceptor to allow aerobic respiration. Initially in return for the ATP through aerobic respiration the α-proteobactia would receive carbohydrates from the host cell

(Andersson 2003). Eventually this endosymbionic relationship, through gene transfer and machinery to import proteins to the mitochondrion, gave rise to the modern mitochondrion found in almost all eukaryotes (Andersson 2003; Ballard et al . 2004;

Emelyanov 2001; Gray et al . 2001; Karp 1999; Lang et al . 1999; Scheffler 2001;

Scheffler 1999; Yang et al . 1985).

1.3 Mitochondrial Genome

Almost all animal mitochondria contain closed circular mitochondrial deoxyribonucleic acid (mtDNA) with the exception of the cnidarian classes Cubozoa,

Scyphozoa and Hydrozoa that have a linear chromosomal mtDNA. The mtDNA of metazoans is typically ~16 kilobases (kb) (15-20kb) (Boore 1999) though this too is defied by some species with the largest animal mitochondrial genome being ~35 kb (from

31-42 kb) belonging to Placopecten magellanicus (Scheffler 1999; Smith and Snyder

2007; Snyder et al . 1987). The smallest mitochondrial DNA is 14 kb and belongs to

Caenorhabditis elegans (Okimoto et al . 1992; Scheffler 1999). There is a tendency for ectotherms to have larger mitochondrial genomes compared to endotherms (Rand 1993). 9

There are typically 37 genes in the mtDNA that are tightly packed and lack introns with the exception of Metridium senile which has group I introns in the COI1 and Nad5 genes

(Beagley et al . 1998). The 13 proteins encoded in the mtDNA are cytochrome oxidase subunits 1-3 (COI 1-3), apocytochrome b (CytB), ATPase subunits 6 and 8 (Atp6 and

Atp8) and NADH dehydrogenase subunits 1-6 and 4L (Nad1-6 and Nad4L). There are the genes that encode the large and small rRNAs (LSUrRNA and SSUrRNA) as well as

22 transfer RNAs (tRNAs) which are used in the transcription of proteins in the mitochondria (Beagley et al . 1998; Scheffler 1999). Though the mitochondrial genome does encode some of the proteins for the ETC, many are also imported from the nuclear genome (Andersson 2003; Scheffler 2001; Scheffler 1999). It is believed that since the endosymbiotic relationship formed, many genes have been lost from the endosymbiont and others have moved to the nucleus (Adams and Palmer 2003; Andersson et al . 1998;

Andersson 2003; Ballard et al . 2004; Bensasson et al . 2001; Emelyanov 2001; Gray et al .

2001; Lang et al . 1999; Lopez et al . 1994; Moritz et al . 1987; Scheffler 2001; Scheffler

1999; von Heijne 1986; Yang et al . 1985).

Gene order of the mitochondrial genome tends to be conserved in many phyla except within Mollusca (Boore 1999). Since the mtDNA mutates 10 times as fast as the nuclear DNA there can be much divergence in the sequence rendering deep phylogenies difficult to deduce. Gene order is thought to be conserved and a useful tool in determining the deep phylogenies (Blanchette 1999). Though gene order would help to resolve some unclear branches for most phyla, it cannot be used in the phylum Mollusca because the gene order changes significantly within the phylum (Boore 1999). 10

Some scallops have unusual features of their mtDNA such as repeat sequences

(Gjetvaj et al . 1992). Placopecten magellanicus has a repeat region of 1.4 kb that is repeated between two to eight times which causes size variation throughout the species and its large mtDNA size (Smith and Snyder 2007; Snyder et al . 1987). This type of repeat has also been seen in other scallops but Placopecten magellanicus has the greatest size variation. Size variation and repeat sequences have been reported in other scallops including Pecten maximus (1.6 kb with 3-7 repeats), Crassadoma gigantea (1 kb with 3-5 repeats), Aequipecten opercularis (~0.4 kb with an unknown number of repeats),

Chlamys hastate (~0.6 kb with an unknown number of repeats) and Chlamys islandica

(1.2 kb with 1-3 repeats) (Gjetvaj et al . 1992). Though this does occur in some scallops it does not occur in all; Argopecten irradians does not contain a repeat sequence (Gjetvaj et al . 1992; Petten and Snyder 2007).

1.4 Secondary Structure and Compensatory Mutation

Secondary structures are found in certain RNA such as transfer tRNA, that fold into structures resembling clover leafs, and rRNA that have three domains and are more complex structures than tRNAs (Karp 1999). tRNAs are usually made up of ~75 nucleotides (nt) whereas rRNAs are usually 1-2 kb. Secondary structures are formed by the RNA folding with itself using hydrogen bonds to form stems (Noller 1984). The normal AU and GC bonds are present as well as unusual UG bonds (Dixon and Hills

1993). The nucleotides that do not form hydrogen bonds form loops, which are typically found at the end of stems but can also be found bulging out in the middle of a stem (this occurs more often in rRNAs than tRNAs) (Karp 1999). 11

Compensatory mutation is defined as a second-site mutation at a distance away from an original mutation required to permit a molecule to continue to function.

Compensatory mutation can be observed in the sequences that make up the stems of rRNAs. An altered sequence at one site only occurs if there is a second alteration that restores base pairing at another site, thereby maintaining secondary structure (Chao and

Poon 2005; Dixon and Hills 1993). For example if in a stem a G mutated to an A, the C that would bond with the original G will mutate to a U to allow the base pairing to continue and therefore the structure maintains stability. It is an important evolutionary process that is not well understood (Chao and Poon 2005).

Dixon and Hills (1993) described 75 classes of mutation that can occur in stems that are stabilized by hydrogen bonded complementary base pairs. Of these, 60 result in disrupting the complementary base pairing, and thus are not ‘compensatory’. Of the remaining 15, there are four ‘one-step’ changes and 11 ‘two-step’ changes that do result in compensatory change. The one-step changes include: A-U to G-U, C-G to U-G, G-C to G-U, U-A to U-G. These changes are made possible by the unusual binding of U and

G and therefore only one member of a base pair changes instead of both. The two step changes include: A-U to C-G, A-U to G-C, A-U to U-A, A-U to U-G, C-G to G-C, C-G to G-U, C-G to U-A, G-C to U-A, G-C to U-G, G-U to U-A and G-U to U-G. In each of these one base changes and therefore the other nucleotide with which it bonds must also change to continue base pair interaction, and therefore two changes are necessary (Dixon and Hills 1993).

The conserved secondary structure exhibited by rRNAs (Noller 1984) has become an important tool when using rRNA sequence for phylogenies. It permits the comparison 12 of functionally equivalent portions of the molecule. Normally any sequence change is considered to be an independent event and therefore is weighted equally to any other change, but compensatory changes observed in the stems of rRNA cannot be thought of as entirely independent. Thus, it is necessary to decide how to weight these changes

(Dixon and Hills 1993). Dixon and Hills (1993) used 1,989 base pairs of the 28S rRNA from: Cryinella lutrensis, Latimeria chalumnae, Xenopus laevis, Rhineura floridana, Mus musculus, Rattus noregicus and Homo sapiens with Drosophilia melanogaster and

Lampetra aepytera as outgroups to determine how to weight the stem nucleotides.

Changes in a base in the loops are weighted as 1.0 since they mutate independently but changes in the stem are weighted less. Compensatory mutation occurs more frequently than by chance but does not always occur and is therefore between 0.5 (always occurs) and 1.0 (never occurs). Dixon and Hills (2003) aligned the sequences to determine the stems from the loops. The stems were analyzed by comparing the phylogeny to previous phylogenies, to obtain the value to weight the stem nucleotides. They found that the stems should be weighted as 0.81 to obtain an accurate depiction of the true phylogeny, but this may vary from species to species (Dixon and Hills 1993).

Dixon and Hills (1993) estimated how compensatory mutation should be weighted in a phylogeny but did not estimate the rate. Chao and Poon (2005) were the first to estimate the rate of compensatory mutation. They used the bacteriophage ФX174 and induced a specific mutation that would decrease the fitness of the bacteriophage. The base pairs that interacted with any altered bases had been previously identified. They induced mutation and looked for phage that had regained function, which would occur either by a back mutation (revert back to wildtype state), or by a second-site, 13 compensatory mutation. They observed that compensatory mutation occurred 67.7% of the time and was more prevalent with more detrimental mutations (Chao and Poon 2005).

The weighting value for compensatory change determined by Dixon and Hills (1993) will be used in the phylogenies reported here.

1.5 Purpose of Study

In this thesis, I report on the success of my efforts to obtain sequence from the mitochondrial DNA of the Iceland scallop, Chlamys islandica . The rRNA gene sequences I obtained are used to determine Chlamys islandica’s phylogenetic relationship within scallops. Sequences obtained for protein coding genes are used to determine the phylogenetic relationship of Chlamys islandica and other scallops within bivalves. The implications of the phylogenies are discussed.

Chapter 2: Sequence Analysis

1. Introduction

In the two billion years since the Rickettsea-like organism was engulfed by a primitive eukaryote, many genes have been lost from the mitochondrial genome (Adams and Palmer 2003; Andersson 2003; Ballard et al . 2004; Emelyanov 2001; Gray et al .

2001; Lopez et al . 1994; Scheffler 2001; Scheffler 1999; Yang et al . 1985). There are three possible outcomes of the gene loss. The first possibility is the gene is lost completely. In this circumstance the gene could become obsolete to the organism or a nuclear gene will replace the function. The next possibility is that the gene transfers to the nuclear genome and becomes a functional gene whose product is imported back into the mitochondria. The final possibility is that the gene attempts to transfer to the nuclear genome but becomes a non-functional pseudogene (Adams and Palmer 2003). Some 14 genes remain in the mitochondrial genome that have not transferred to the nuclear genome. Some believe that this is due to high hydrophobicity of the proteins that would make import back into the mitochondria unfavourable (Adams and Palmer 2003). Since

COI1 and CytB are genes found in all mitochondrial genomes and they are highly hydrophobic they support this hypothesis (Adams and Palmer 2003). The hydrophobicity hypothesis does not explain all the genes still found in the mtDNA. Another hypothesis is that the non-standard code for the mitochondrial genome could prevent further functional transfers to the nuclear genome. Since the mitochondrial genome does not follow the standard genetic code this could cause amino acids to be incorrect if they are in the nuclear genome that uses the standard genetic code. This could also cause premature stop codons making them non-functional (Adams and Palmer 2003).

A pseudogene is a sequence which resembles a gene but is not the actual gene

(Bensasson et al . 2001). If a pseudogene sequence is inadvertently used in a phylogeny it can produce inaccuracies as they can have a higher rate of mutation than the functional gene because there is no selective pressure. Mitochondrial pseudogenes found in the nuclear genome are called nuclear mitochondrial pseudogenes or Numts (Bensasson et al .

2001; Lopez et al . 1994). When a Numt is present for the region of interest in the mitochondria, a PCR primer could hybridize and amplify the Numt as well as the gene or in preference to the gene of interest (Collura and Stewart 1995; Sorenson and Quinn

1998; Zhang and Hewitt 1996). If present and undetected they can lead to misleading phylogenies (Sorenson and Quinn 1998). 15

In this chapter, the sequences for partial sequence of mitochondrial DNA genes along with their hypothetical amino acid sequence are examined. The inference of possible gene order for two genes is examined.

2. Materials and methods

2.1 Sample Collection

Samples of Chlamys islandica spat were obtained from Mahone Bay, Nova

Scotia, through Duncan Bates in June 2004.

2.2 DNA isolation

DNA isolation was carried out by Mark Petten as outlined in Petten and Snyder

(2007). Five spat were ground in liquid nitrogen in a microcentrifuge tube using a micropestle. Total DNA was isolated from five spat using the CTAB method. The 2%

CTAB DNA extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris

HCl pH 8.0) was prewarmed to 67 oC before addition to the ground spat. A volume of

800 µl was added to the ground individuals and they were incubated at 67 oC for 30 minutes. The sample was then centrifuged at 4000 rpm for 5 minutes to remove the crushed shells and other debris. The supernatant was removed and placed in a clean tube and 500 µl of 24:1 chloroform/isoamylalcohol was added. The mixture was inverted 50 times to ensure complete mixing rather than vortexing to prevent shearing of DNA. The mixture was centrifuged at 11000 rpm for 15 minutes. The upper aqueous phase was transferred to a clean tube and the bottom layer was discarded. To the aqueous phase

325 µl of cold isopropanol was added and mixed thoroughly to precipitate the DNA. The solution was left overnight at -20 oC to increase the yield. The following day, the mixture 16 was centrifuged at 6000 rpm for five minutes. All of the liquid was removed from the pelleted DNA. The pellet was washed with 550 µl of 70% ethanol and centrifuged at

6000 rpm for 5 minutes. A second washing step was performed with 550 µl of 100% ethanol and again centrifuged at 6000 rpm for five minutes. The ethanol was removed and the pellet air dried. The pelleted DNA was resuspended in 30 µl of molecular grade water (Gibco) (Petten and Snyder 2007).

2.3 Gene Amplification

2.3.1 Primers

The sequence for universal primers for COI (Lco1490 and Hco2198) were obtained from Kocher et al . (1989). Another set of primers for COI (COXF and COXR) were designed using the sequence from Placopecten magellanicus , Argopecten irradians and Mizuhopecten yessenosis because of the suspicion that the universal COI primers were amplifying a pseudogene of COI. From here onwards the amplification performed by the universal primers will be referred to as COIa and the amplification performed by the other set of primers COIb. The sequences for universal primers for 12S were obtained from Barucca et al. (2004) and the sequences for universal primers for CytB from Boore and Brown (2000). Primers sequences were designed for Atp6, 16S and

Nad1 by comparison of Placopecten magellanicus and Argopecten irradians sequence.

Primers sequences were designed for Nad3 and Nad4 by comparing the sequences from

Mizuhopecten yessenosis, Placopecten magellanicus and Argopecten irradians. Primers design was performed using the Primer3 webserver (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi). Primers used for individual gene amplification can be found in Table 1. All primers used in this study can be found in Appendix 1.

Table 1. Primers used in the amplification of mitochondrial genes for PCR. Primer Target Gene Primer Sequence (5’ 3’) LCO1490 COI GGTCAACAAATCATAAAGATATTGG Hco2198 COI TAAACTTCAGGGTGACCAAAAAATCA COXF COI GTTYTWATTGGDGGGTTTGG* COXR COI TGMCCCCAYACAATAAAVCC* 16SL 16S TCGTACCTTTTGCATCATCATGG 16SR 16S TTTTGCCGAGTTCCTTTAGC 12S-F 12S AGACATGGATTAGATACCC 12S-R 12S ACCCCTACCTTGTTACGACTT ATP6L Atp6 TTRAGGACTTAYTTTGATCAGTTG* ATP6H Atp6 GCCAAAAKAATATGACCTCAM* cobF424 CytB GGWTAYGTWYTWCCWTGRGGWCARAT* cobR876 CytB GCRTAWGCRAAWARRAARTAYCAYTCWGG* ND1F Nad1 TTTGAGCGRAARGTKTTRSC* ND1R Nad1 CATAWARRCCSCCCCAAAAA* ND3F Nad3 GGKTBMGGGATTCYCARAAG* ND3R Nad3 AAWGAYTCHACYTCAAABACCA* ND4F Nad4 GTTTTGGYTGGGVGCTYTWAT* ND4R Nad4 CTCAACRTGMGCYTTAGTYAACC* *R=A or G; Y=C or T; W=A or T; K=G or T; M=A or C; S=G or C; B=G, T or C; H= A, T or C; V=G, A or C; D=G, A or T

2.3.2 Polymerase Chain Reaction

The PCR was first carried out for the primers in Table 1 in a 25 µl reaction to confirm the reaction conditions were optimal, and then the PCR was performed in a 50 µl reaction for gel extraction and purification. This was done to ensure that there was a high enough concentration of DNA for sequencing. All reactions were performed in a MJ

Research PTC-200 thermal cycler. Reaction conditions were modified for each primer pair as shown in Table 2 and Table 3. The Taq DNA polymerase, 5X buffer, 25 mM

MgCl2 and dNTP’s were obtained from Promega.

Table 2. The optimal PCR conditions for each gene Reagents Target Gene COIb COIa CytB 16S 12S Atp6 Nad1 Nad3 Nad4 5X buffer 1X 1X 1X 1X 1X 1X 1X 1X 1X 25mM 2.5mM 2.5mM 2.5mM 2.5mM 2.5mM 2.5mM 2.5mM 2.5mM 2.5mM MgCl 2 2.5mM 2.5mM 250µM 250µM 250µM 250µM 250µM 250µM 250µM 250µM dNTPs 10µM 500µM 500µM 500µM 500µM 1000µ 500µM 500µM 500µM 500µM Primer 1 M 10µM 500µM 500µM 500µM 500µM 500µM 500µM 500µM 500µM 500µM Primer 2 Taq 5U/µl 0.15U/µ 0.1U/µl 0.05U/µ 0.1U/µl 0.05U/µ 0.1U/µl 0.1U/µl 0.1U/µl 0.1U/µl l l l DNA 2ng/µl 2ng/µl 2ng/µl 2ng/µl 2ng/µl 2ng/µl 2ng/µl 2ng/µl 2ng/µl 100ng/µl ddH 2O *q.s to final volume

Table 3. PCR cycling parameters for each gene Cycling Step Target Gene COIb COIa CytB 16S 12S Atp6 Nad1 Nad4 Nad3 Initial 95 oC, 95 oC, 95 oC, 94 oC, 94 oC, 94 oC, 94 oC, 95 oC, 95 oC, Denaturation 1:00 1:00 1:00 2:00 1:00 2:00 2:00 1:00 1:00 Denaturation 95 oC, 95 oC, 95 oC, 94 oC, 94 oC, 94 oC, 94 oC, 95 oC, 95 oC, 1:00 1:00 1:00 1:00 0:30 1:00 1:00 1:00 1:00 Annealing 47 oC, 45 oC, 50 oC, 47 oC, 52 oC, 47 oC, 47 oC, 45 oC, 47 oC, 1:00 1:00 1:00 1:00 0:30 1:00 1:00 1:00 1:00 Extension 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 1:30 1:30 1:30 2:00 1:00 2:00 2:00 1:30 1:30 Number of 36 36 36 35 30 35 35 36 36 Cycles Final 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, 72 oC, Extension 2:20 2:20 7:00 7:00 2:00 7:00 7:00 2:20 2:20

2.3.3 Gel electrophoresis, band visualization and excision

Each PCR reaction was run out on a 1% TAE agarose gel containing ethidium bromide and in 1X TAE. The Hind III digested λ DNA ladder (Invitrogen) was added to the first well of each gel. The gels were allowed to run at 110V for 30 to 40 minutes.

The gels were visualized under UV light with a Bio-Rad Gel Doc 2000 system. Bands were excised from the gel using a scalpel and tweezers and placed in 1.5 mL tubes. The excised gel was kept at 4 oC until removal of DNA from the gel.

2.3.4 Isolation of DNA from agarose gel

The DNA was isolated from the gel using a QIAEX II Gel Extraction Kit

(Qiagen). The excised pieces of gels were weighed and QG buffer of three times the volume of their weight was added (i.e. 300 µl of QG Buffer to 100 mg of gel). These were placed in a 50 oC water bath for 10 minutes or until gel was dissolved. These tubes were vortexed every 2 to 3 minutes. A volume of isopropanol equal to the weight of the gel was then added (i.e. 100 µl of isopropanol to 100 mg of gel). The tubes were inverted

3 to 5 times to mix. The solution was then transferred from the 1.5 mL tube to a spin 20 column (Qiagen) 800 µl at a time and centrifuged at 13000 rpm for 1 minute. If the solution exceeded 800 µl, the first 800 µl was added and centrifuged then the remainder was added and centrifuged at 13000 rpm for 1 minute. The flow through was discarded.

Another 500 µl of QG buffer was added and centrifuged for 1 minute at 13000 rpm. The flow through was discarded. 750 µl of PE buffer was added and allowed to sit 5 minutes before being centrifuged at 13000 rpm for 1 minute. The flow through was discarded then centrifuged for another 1 minute at 13000 rpm. This flow through was also discarded. The spin columns were then placed in 1.5 ml Eppendorf tube and 30 µl water was added and allowed to stand for 1 minute before being centrifuged for 2 minutes at

13000 rpm. Another 30 µl was added and allowed to stand for 1 minute and centrifuged at 13000 rpm for 2 minutes. A small portion of the isolated DNA was run on a 2% agarose TAE gel containing ethidium bromide in 1X TAE buffer for approximately 45 minutes at 110V with the DNA ladder, low range (Fermentas) in the first well as a marker to determine concentration. The remaining sample was stored at -20 oC.

2.3.5 DNA sequencing

Sequencing of the PCR products was done by Dr. Steve Miller at Florida State

University Sequencing Facility. The sequencing used Big-Dye terminator chemistry and analysis that was performed by an Applied Biosystems 3100 Genetic Analyzer with

Capillary Electrophoresis. The same primers used in the PCR were also used in the sequencing. The results were provided in an AB1 format and analyzed using Bioedit 7.0

(Hall 1999).

2.3.6 Sequence comparison

Once the sequence was obtained and edited it was submitted to Basic Local

Alignment Search Tool (BLAST) (Altschul et al . 1997) on the National Center for

Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov), to ensure that the gene amplified was indeed the correct gene. Once the genes were confirmed (i.e. were similar to the same gene in other organisms) the whole genome amplification was attempted.

2.3.7 Amino acid sequence

The amino acid sequences were deduced by comparison to the reading frames of other scallops to determine the reading frame. Mega 3.1 (Kumar et al . 2004) translated the DNA sequence to the amino acid sequence once the reading frame was determined.

This used the standard genetic code. The amino acid sequence was then edited using the invertebrate mitochondrial code for each codon.

2.4 Whole genome amplification

Long accurate PCR (LAPCR) can be used to amplify the mitochondrial genome.

It uses a special DNA polymerase that contains extra enzymes (Klentaq1 and 5, Pfu ,

KTLA-64 and Amplitaq) that allows for amplification of larger fragments (up to 35kb) of

DNA in a single PCR. These enzymes also increase the fidelity of the DNA sequenced

(Barnes 1994). Primers used to amplify genes are oriented so that they ‘point’ inward, toward the center of the gene. In contrast, for whole genome amplification, primers are designed to ‘point’ outward, away from the gene and toward neighboring sequences.

This theoretically permits the acquisition of all portions of the genome between two genes. If the sequence between the genes is determined then the whole genome can be 22 sequenced and then aligned. LAPCR allows the amplification of the sequence between genes and therefore is used for whole genome amplification.

2.4.1 Primer design

Primers were designed using Primer3 0.3.0 (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi) from the sequences obtained from the PCRs. They were designed to be approximately 30 base pairs each and have a GC% of 30 % to 40 % (with the minimum being 25% if necessary). The primers used in the successful LAPCR can be found in Table 4. All primers used in this study can be found in Appendix 1.

Table 4. Primers used in successful LAPCR. Primer Target Primer Sequence (5’ 3’) 12S-ND1L 12S-Nad1 ACTTAGGAGTAAGGGGAGATTAATATGCTT 12S-ND1R 12S-Nad1 ATAAAACAGGGACCTAGAAAAAACAACAC

2.4.2 Long accurate polymerase chain reaction

The LAPCR were carried out using the primers designed from the sequences obtained through PCR. The reaction was carried out at 25 µl to determine if the primer pairs and reaction conditions were optimal. If the 25 µL reaction was successful a 50 µL was performed for band extraction and sequencing. The reaction conditions were 1X iProof HF Buffer, 200 µM each dNTP, 0.2 µM each primer, 25 ng DNA template, sterile water (to final volume), 0.2 U/µl iProof high fidelity DNA polymerase (added in the order listed). The iProof High fidelity DNA polymerase, iProof HF buffer and MgCl 2 were obtained from Bio-Rad. The dNTP solution was obtained through Takara. The cycling parameter were 98 oC for 30 seconds for initial denaturation, 98 oC for five seconds for denaturation, 67 oC for 15 seconds for annealing, 72 oC for two minutes and 30 23 seconds for extension for 40 cycles and 72 oC for final extension for 10 minutes. The reactions were stored at 4 oC until they were analyzed through gel electrophoresis.

2.4.3 Gel electrophoresis, band visualization and excision

The electrophoresis was done as described above in Section 2.3.3 with the exceptions that commassie blue dye was added to the samples before they were loaded and allowed to run for one hour at 110V.

2.4.4 Isolation of DNA from agarose gel

The isolation was done as described above in Section 2.3.4 with the exception that a small portion of the DNA was run on a 1% agarose TAE gel containing ethidium bromide in TAE for 45 minutes with the Hind III digested λ DNA ladder (Invitrogen) in the first lane to determine concentration.

2.4.5 DNA sequencing

The sequencing was done as described in Section 2.3.5.

2.4.6 Sequence comparison

Once the sequence was edited it was submitted to the BLAST tool (Altschul et al .

1997) on the NCBI website to confirm that is was of mitochondrial origin. The sequence was then submitted to tRNAscan-SE (Lowe and Eddy 1997) and DOGMA (Wyman et al .

2004) to search for possible tRNAs. tRNAs were also searched for visually by looking for the 5 ’3’ sequence: ‘ANNRYNNNNNNYT(anticodon)RN’ as outlined by Smith and Snyder (2007).

3. Results and Discussion

3.1 Gene amplification

The gel electrophoresis images, DNA sequence and the deduced amino acid sequence for the protein coding genes are presented below. PCR was used to successfully amplify the genes COIa and b, CytB, 12S, 16S, Atp6, Nad1, Nad3, and

Nad4. Each PCR produced a single band except for sample Chlamys B for the 16S which produced 2 bands. Both were excised and sequenced. COIb had two bands in which the larger band was sequenced since it was the appropriate size. The images for each of these gels are seen in Figures 1-8. The sequences were then confirmed to be from mitochondrial origin and the correct gene using the BLAST tool (Altschul et al . 1997) on the NCBI website. The sequences were then used to design ‘outward pointing’ primers for the LAPCRs, with the intent of using the primers to amplify intergeneic regions.

Figure 1. 16S amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the 50X concentration negative control, lane 5 is the Chlamys A 100X concentration, lane 6 is the Chlamys B 100X concentration and lane 7 is the 100X concentration negative control. The common band among all is approximately 200 bp and the 2 nd band on Chlamys B is 300 bp.

Figure 2. CytB and 12S amplifications. In lane 1 Hind III digested λ DNA ladder, in lane 2 is Chlamys A for the CytB amplification, in lane 3 is the Chlamys B for the CytB amplification and in lane 4 is the CytB negative control. The band was approximately 400 bp. In lane 5 is the Chlamys A for the 12S amplification and in lane 6 is the Chlamys B for 12S amplification and lane 7 is the negative control for 12S. The bands are approximately 450 bp.

Figure 3. COIb amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the 50X concentration negative control, lane 5 is the Chlamys A 100X concentration, lane 6 is the Chlamys B 100X concentration and lane 7 is the 100X concentration negative control. The largest band was isolated and is approximately 500 bp.

Figure 4. COIa amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the 50X concentration negative control, lane 5 is the Chlamys A 100X concentration, lane 6 is the Chlamys B 100X concentration and lane 7 is the 100X concentration negative control. The bands are approximately 300 bp.

Figure 5. Nad1 amplification. In lane 1 Hind III digested λ DNA ladder, in lane 2 is Chlamys A 50 X concentration, in lane 3 is Chlamys B 50X concentration, in lane 4 is the negative control for 50X concentration, in lane 5 is Chlamys A 100X concentration, in lane 6 is Chlamys B 100X concentration and in lane 7 is the negative control for 100X concentration. All products were approximately 300 bp. 2 nd row was an attempt to amplify COI3 which did not amplify the proper gene.

Figure 6. Nad3 amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the 50X concentration negative control, lane 5 is the Chlamys A 100X concentration, lane 6 is the Chlamys B 100X concentration and lane 7 is the 100X concentration negative control. All are approximately 200 bp.

Figure 7. Nad4 amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the 50X concentration negative control, lane 5 is a failed attempt with Chlamys A 100X concentration, lane 6 is a failed attempt with Chlamys B 100X concentration and lane 7 is the 100X concentration negative control. The bands were approximately 400 bp.

Figure 8. Atp6 amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 is Chlamys A 50X concentration, lane 3 is Chlamys B 50X concentration, lane 4 is the negative control for 50X concentration, lane 5 is Chlamys A 100X concentration, lane 6 is Chlamys B 100X concentration and lane 7 is the negative control for 100X concentration. All products were approximately 400 bp.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 CTTATTCGTA CCTTTTGCAT CATGGAGCTG GTATCAGGAC GMCTGACACA

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 CAGATTCAGT ACCAAGGAGG WACCCTTGTG TATATGTRTT ACAGCCAGTA

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 ACCATGTCAT TGTAGGGATG ACCAAACACA TCTCCAAATT TACCACAGAT

....|....| ....|....| ....|....| ....|....| ....|. 160 170 180 190 GGTAAAATKG TGTGTACTAC TATGGCTAAA GGAACTCGGC AAAAAA

Figure 9. The edited partial sequence for the 16S (LSUrRNA) of 196 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TTATTACCGA CGCAAACTTT GCTATGAGGC AGCTGCTTGG GTACTACGAG

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 CGTGTGCTTA AAACTCAAAG AACTTGGCGG CTCGTTAACT ACCTAGGGGA

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 ATATGCGCCT TAATCCGATG ATCCGCGTAG CATCTTACTG TACCTTGAAA

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 AAGAACAGCT GGTGTATTGC CGTCGTCAGC CTGTTGTTCG AGCAAGGAGA

....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 AACAGGCCCA AGGGAACTGG CGATTTGTCG ACAGGATCCA TAAAGTCAGG

....|....| ....|....| ....|....| ....|....| ....|....| 260 270 280 290 300 TCGAAATACT GCCTATGGTA CGAGGGAGTG GGTATTACAA TTCAAAATTC

....|....| ....|....| ....|....| ....|....| ....|....| 310 320 330 340 350 GAAATACGGA GCTTGGAAGA ACTGTGAAAT CTCCAGGTGA AGGTGGACTT

....|....| ....|....| ....|....| ....|....| ....|....| 360 370 380 390 400 AGGAGTAAGG GGAGATTAAT ATGCTTCCCT GAACGTGAAT CTAACTTGTG

....|....| .. 410 TACAAACCGC CC

Figure 10. The edited partial sequence for the 12S (SSUrRNA) of 412 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TTCGCCCTTT ATATGGTGGT GGTGTCTCCT TTTATCGAGC AGGGGAGTGG

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 AACAGGATGA ACCATATATC CTCCTTTGTC TTCTACTCCA TACCAGGGGG

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 ATATTTGCAC AGACATAGTA ATCTTAGGGT TGCATTTGTC TGGGGTAAGC

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 TCTACTGCTG GGGCGATTAG TTATCTGGTC ACTTTTTTAA ACATACGCGG

....|....| ....|....| ....|....| .... 210 220 230 AAAGTCCTAT ANGGCGGAGT TTTGTCCGCC CTTC

Figure 11. The edited partial sequence of COIb of 234 nucleotides. This is thought to be the actual gene for COI

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TGGTCAACAA ATCATAAAGA TATTGGTCAG AGATTTTTCA AAATGTTTAT

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 CTTCCACAGG TTTTTTCAAT TATGTATTTG CCATTTCTAC CTTCTGATAT

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 AATTGATGTT CATTATCGTA TTGCACATAA CTCTATATTT ACGATGGAGA

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 AGTTAAAGAA AATTGGCAAA GTAGATAGCG ATACATGTCT GTTGTGCGAC

....|....| ....|....| ....|....| ....|....| .. 210 220 230 240 TCTGAGACGG AAACTTTACT GCATTTGTTT GTAGATTGTT CA

Figure 12. The edited partial sequence of COIa of 242 nucleotides. This is suspected to be a pseudogene.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 GGTTACTGCC ATCCCTGTTG TGGGGAAGGA TGTTGCTATG TGGGTGTGGG

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 GCGGGTATGC TGTTGGAAAC GCTACCCTTA AGCGTTTGTT TAGAATCCAT

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 TTTCTGCTTC CTTTTATCAT CCTGGCGGTT TTTTTTCTTC ACTTAACTTT

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 TCTTCATGAG AGGGGGTCGG GAAATCCTTT AGGTGTGGAG ACGGACTGTA

....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 ATTTAGTTCG ATTTCATACG TTTTTTATGG TGAAAGATTT GGTGGGGGTG

....|....| ....|....| ....|....| ....|....| ....|.. 260 270 280 290 ATGGGGGCTT GTAGGCTTTT GTGCTATGTA GTCTCTCGAC ATCCCTA

Figure 13. The edited partial sequence of the CytB gene of 287 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 CATCCCAAGG TSRAAAAGGT CCAGAAATGG TAGGTTGGTA CGGAATTCTT

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 CAACCATTTG CGGACGGAAT CAAGYTGTTT AKAAAARAGT ATTTTACTCC

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 CAGGGACTST AACCCAGYGK TGTTTTTTCT AGGTCCCTGT TTTATGCTGT

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 TCCATTCTTT TGTTCTGTGG GGGTGTAGCC CGGGGGTTTG SGGGTGTGGA

....|....| ....|....| ... 210 220 RTTTATTTTT TTTGGGGCGG GCT

Figure 14. The edited partial sequence of the Nad1 gene of 223 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TGATCTTTTC GGCTCTGCTC GAGCTCCTTT TTCCGTGCGA TTTTACATGG

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 TAGCTATTTT ATTTCTGGTG TTTGAGGTGG AGTCATTGAG GCKCGGGATT

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 CTCAGAAGGC TTCTCCCTAC GAGTGTGGTT TTGATCCTTT CGGCTCTGCT

....|....| ....|.. 160 CGAGCTCCTT TTCCGTT

Figure 15. The partial edited sequence of the Nad3 gene of 167 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TGTGTATTTG GGGGGTGGTC CTTTGTTGTT TCTTAGCTTT TTGATCTCAC

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 AGCTTTCTTT GGTTTTATAT TTTCTTTGAG CTTTCTTTAG TTCCTGTTTT

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 TGTCTTAATT CTTCGATGAG GAATTCAGCC TGAGCGTATT AGGGCTGTGT

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 TTTATTTAAT GCTGTACACT CTTACAGGAT CTCTTCCCTT TTTGCTATTT

....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 ATTCTGTCAT GTTTTTGAAT TTACGGGTCT TTTTTTATGG GGTTTAGTTG

....|....| ....|....| ....|....| ....|....| ....|....| 260 270 280 290 300 GGATTTACTA AGAAGAATGG GGTATTGGGG TTGTGTTGGT GTGATGGTTT

....|....| ....|....| ....|....| ....|....| 310 320 330 340 TTTTTATCAA GATACCTTGT TATCCATTTC ACTTGTGGTT

Figure 16. The edited partial sequence of the Nad4 gene of 340 nucleotides.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TTTTTTGGCT AGTTTAGTTT AGACAAMAAT TTGGATTACA CAGAGTGTAC

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 CATAGATATA AGGGCGTTAT TTAATAAAGT TAACATCTTA TTAACTCCTA

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 TYGAACCRTG TAGAGAAAAC CAACCAATTT WGGTTTGATG GCTTACCTTT

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 AATTTGTTGA TAATTCARTC ATTATCATGC AATMRGCAAT GTCTAAAATA

....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 TGARATATAT TCAAGTGSYA AACTGAAGCT TTGTACAATG TATTAGYCCA

....|....| ....|....| ....|....| ....|. 260 270 280 AAGGTGTGGG ATATATTTGG TGGTTGATTG AAGTTT

Figure 17. The edited partial sequence of the Atp6 gene of 286 nucleotides.

Figures 9-17 are the sequences obtained from Chlamys islandica for each gene.

There are two sequences presented for COI, a and b. COIa was amplified using the universal primers designed by Kocher (1989) and COIb was amplified from designed primers (see material and methods, Section 2.3.1). COIa was sequenced first and did appear to be the COI gene because when submitted to BLAST tool (Altschul et al . 1997) on the NBCI website it was similar to the COI gene in Achelia bituberculata which is an arthropod. When the sequence was closely examined in comparison to the other scallop species, Argopecten irradians, Mizuhopecten yessenosis and Placopecten magellanicus there was some similarity but much divergence in comparison to the other three scallops.

The phylogeny produced using this sequence did not show the typical grouping

(discussed in more detail in Chapter 2). The amino acid sequence for the COIa contains stop codons which also indicates it is a pseudogene. It was due to these dissimilarities 38 which provoked designing new primers because of the suspicion of a possible pseudogene. The designed primers were used to amplify the COI gene in a different conserved region than the universal primers. When the sequence for COIb was obtained and submitted the BLAST tool (Altschul et al . 1997) on the NCBI website it was shown to be similar to other scallop COI genes. This supported the hypothesis that COIa was a pseudogene that would most probably have migrated to the nuclear genome . Once it reached the nuclear genome it would have become nonfunctional because either COI is too hydrophobic to import into the mitochondrion or the difference between the mitochondrial genetic code and standard genetic code would have changed the amino acid sequence therefore changing the protein or caused premature stop codons (Lopez et al . 1994). Once it became nonfunctional then its mutation rate would have been higher than in the actual gene because there would not be selection to conserve it, which would explain the divergence. Since it was amplified by the universal primers this would indicate that their recognition site would probably still be intact and would have out- competed the mitochondrial COI gene in the amplification. When the designed primers

(COXF and COXR) were used to amplify the COI gene, the mitochondrial gene (i.e. the actual gene) was amplified.

FALYMVVVSPFIEQGSGTGWTMYPPLSSTPYQGDICTDMVILGLHL SGVSSTAGAIIYLVTFLNMRGKSYKAEFCPPF

Figure 18. The hypothetical amino acid sequence for COIb gene.

The hypothetical amino acid sequence for the COIb gene is depicted in Figure 18.

This is predicted due to alignment of the sequence to other scallop COI genes and where the coding would start. Since it is not the completed gene the codons could be off by a base or two causing the entire amino acid sequence to change. Since the complete gene 39 is not sequenced this is the hypothetical amino acid sequence for this portion of the gene.

This as well as the other amino acid sequences presented below are based on the invertebrate mitochondrial genetic code.

GFFNYVFAISTFWYNWCSLSYCT*LYIYDGEVKENWQSS*RYMSVV RLWDGNFTAFVL*IV

Figure 19. The hypothetical amino acid sequence for COIa gene. The * indicates stop codons.

The hypothetical amino acid sequence for COIa is depicted in Figure 19. There are three stop codons within the amino acid sequence which supports the assertion that this is a pseudogene. A coding gene does not have stop codons within the gene.

VTAIPVVGKDVAMWVWGGYAVGNATLKRLFSIHFLLPFIILAV FFLHLTFLHESGSGNPLGVETDCNLVRFHTFFMVKDLVGVMG ACSLLCYVVSRHP

Figure 20. The hypothetical amino acid sequence for CytB gene.

The hypothetical amino acid sequence for CytB is depicted in Figure 20. This again is hypothetical and the proper amino acid sequence should be determined when the full gene sequence is available.

SQG? 1KGPEMVGWYGILQPFADGIKLF? 2K? 3YFTPSD? 4NP? 5?6FFLGP CFMLFHSFVLWGCSPGV? 7GCG? 8YFFWGG

Figure 21. The hypothetical amino acid sequence for Nad1 gene. The ?’s are where there are more than one possible amino acid based on the sequence.

The amino acid sequence for Nad1 is depicted in Figure 21. There are eight amino acids that could not be assigned because of ambiguity in the identity of the base.

The symbols R, Y and K, for example, are used to indicate ‘purine’, ‘pyrimidine’ or ‘G or

T’ when sequencing is not clear. For ? 1 the codon is SRA where S could be C or G and R could be A or G. This leaves four possible amino acids: Q (CAA), R (CGA), E (GAA) or

G (GGA). For ? 2 the codon is AKA where K could be G or T. Therefore there are two 40 possibilities: S (AGA) or M (ATA). For ? 3 the codon is RAG which means the two possible amino acids are K (AAG) and E (GAG). For ? 4 the codon is TST which means the two possible amino acids are S (TCT) or C (TGT). For ? 5 the codon is GYG where Y can be C or T. Therefore the two possibilities are: A (GCG) or V (GTG). For ? 6 the codon is KTG giving the possibilities of V (GTG) or L (TTG). For ? 7 the codon is TGS giving the possibilities of C (TGC) or W (TGG). For ? 8 the codon is RTT which gives the possibilities of I (ATT) or V (GTT). Since the electropherogram was not able to distinguish among the possible bases at each of these positions the amino acids can not be determined, but are narrowed down to the possibilities above. This amino acid sequence is based on the alignment of other sequences and could also be incorrect causing there to be more ambiguities. Since many of the symbols are found in the second base of the codons, this leads to there being more than one possible amino acid. Sometimes if the symbol is in the third position it can be the same amino acid because of the wobble hypothesis. The wobble hypothesis allows more than one codon to code for a particular tRNA, but the wobble position is always the third position (Karp 1999). This could indicate that the amino acid sequence is shifted by one base pair but this cannot be confirmed until the full gene is sequenced. It could also be due to the sequence being ambiguous at these positions.

DLFGSARAPFSVRFYMVAILFLVFEVESLS?GILSSLLPTSVVLIL SALLELLFR

Figure 22. The hypothetical amino acid sequence for Nad3 gene. The ? is undetermined amino acid.

The hypothetical amino acid sequence for Nad3 is depicted in Figure 22. The ? is indistinguishable because the codon is CKC where the K could be G or T. Therefore there are two possible amino acids for this position, either L (CTC) or R (CGC). 41

CIWGVVLCCFLAFWSHSFLWFYIFFELSLVPVFVLILRWGIQPERISAV FYLMLYTLTGSLPFLLFILSCFWIYGSFFMGFSWDLLSSMGYWGCVG VMVFFIKMPCYPFHLW

Figure 23. The hypothetical amino acid sequence for Nad4 gene.

The hypothetical amino acid sequence for Nad4 is depicted in Figure 23. This is based on alignment with other Nad4 sequences.

FLASLV*T? 1IWITQSVP*M*GRYLMKLTSY*LL? 2N? 3VEKTNQF? 4FDG LPLICW*F? 5HYHA? 6?7NV*NM? 8YIQV? 9NWSFVQCISPKVWDMFGGW LKF

Figure 24. The hypothetical amino acid sequence for ATP6 gene. The * indicates a stop codon and ? are undistinguishable amino acids.

The hypothetical amino acid sequence for Atp6 is depicted in Figure 24. The sequence contains six stop codons and nine indistinguishable amino acids. For the indistinguishable nucleotides the ? 1 has the codon is AMA where M can be A or C.

Therefore the possible amino acids are K (AAA) or T (ACA). For ? 2 the codon is TYG where Y could be C or T. Therefore the possible amino acids can be S (TCG) or L

(TTG). For ? 3 the codon is CRT where R can be A or G. Therefore the possible amino acids are H (CAT) or R (CGT). For ? 4 the codon is WGG where W can be A or T.

Therefore the possible amino acids are S (AGG) or W (TGG). For ? 5 the codon is ART and the possible amino acids are N (AAT) or S (AGT). For ? 6 the codon is ATM, and therefore the possible amino acids are M (ATA) or I (ATC). For ? 7 the codon is RGC, and therefore the possible amino acids are S (AGC) or G (GGC). For ? 8 the codon is

ARA, and therefore the possible amino acids are K (AAA) or S (AGA). For ? 9 the codon is SYA where S can be C or G and Y can be C or T, therefore there are four possible amino acids. The possibilities are: P (CCA), L (CTA), A (GCA) or V (GTA). Since there are stop codons within the sequence, it is a pseudogene. Therefore the ambiguity 42 could be caused by different mutations in the sample because there is no selective pressure to keep the sequence the same. Since there are six stop codons within in the sequence it would indicate this gene is a pseudogene. The stop codons could also be due to the amino acid sequence being incorrect if the bases should be shifted. The phylogenetic analysis also gives an indication of a pseudogene (to be discussed in

Chapter 2).

3.2 Amplification of long accurate PCR fragments

LAPCR was used to attempt the amplification of the whole genome. One reaction has worked in high enough yield to sequence. This reaction yielded a small fragment of approximately 500bp and is shown in Figure 25.

Figure 25. 12S-Nad1 amplification. In lane 1 Hind III digested λ DNA ladder, lane 2 Chlamys A 100X concentration, lane 3 Chlamys B 100X concentration, lane 4 is the negative control. The brightest bands were excised and are approximately 500 bp.

....|....| ....|....| ....|....| ....|....| ....|....| 10 20 30 40 50 TCGTAACAAG GTAGGGGTAC TGGAAGGTGC GCCCTGACAA ACAGAAGTGG

....|....| ....|....| ....|....| ....|....| ....|....| 60 70 80 90 100 GGGGAAGGCT GGGAGGAATC TCAATGGTAG TAGTTTACTG CCTTCCCCTA

....|....| ....|....| ....|....| ....|....| ....|....| 110 120 130 140 150 TGTGTGGTAT GAAAGTAAGC TATTATGCTC RATGTTTTGC TTGGCAAGTG

....|....| ....|....| ....|....| ....|....| ....|....| 160 170 180 190 200 TGTCTCTTCT CTTTTTTGGG GGCTTTGCTT CTGGGAGAAG ATATATATGT

....|....| ....|....| ....|....| ....|....| ....|....| 210 220 230 240 250 TGTAGGGTGG GAGTTNTTNC GGTATAGAAG CTTGCTTACG GGAACAGGTT

....|....| ....|....| ....|....| ....|....| ....|....| 260 270 280 290 300 TTTTTTGATC TWGTCGGGCT TATTTTTCAG GGGAKGTTTT TTTAGTTTCT

....|....| ....|....| ....|....| ....|....| ....|....| 310 320 330 340 350 TGGTGTGTTT TTAAGTTTAG GGATTTTTAT ATGCGGGGTG AGCCGTACAG

Figure 26. The edited sequence for the fragment 12S-Nad1 of 352 nucleotides.

The edited sequence for the fragment from 12S to Nad1 yielded 352bp (Figure

26). The sequence does not overlap with the herein reported sequence for 12S or Nad1 as would be expected because the primers are each part of one or the other of those genes.

The sequence did show similarity to Mizuhopecten yessenosis mitochondrial genome using the BLAST tool (Altschul et al . 1997) on the NCBI website. Therefore it is from the mitochondrial genome. Since the primers used to amplify this fragment are from the sequence of 12S and Nad1 it is assumed to be the sequence that separates those two genes. This is not confirmed since it does not overlap either of the sequences. When the sequence was submitted to DOGMA (Wyman et al . 2004) the bases 1-41 corresponded to 45 the 12S gene. Since it does not align with the 12S sequence in Figure 10, it is probably the end of the 12S gene which is not included in the partial sequence obtained. There were no tRNAs identified by DOGMA or tRNAscan-SE. When the sequence was searched visually, one possible tRNA was found. Bases 74-91 followed the sequence in

Section 2.4.6. The possible tRNA structure is found between 49 and 131 and would be trnS . This is based on the anitcodon present and possible base pairing for the secondary structure performed manually and drawn in SmartDraw. Figure 27 depicts the putative secondary structure using the bases 49-131. This has been drawn manually and therefore there is possible error in this secondary structure. The T’s in the sequence have been changed to U’s to represent the tRNA which would contain uracil in place of thymine

(Karp 1999). Assuming that it bridges the two genes, then the gene order would be 12S- trnS -Nad1. These are the only genes that can be ordered in the mtDNA molecule.

trnS Figure 27. Putative secondary structure for the tRNA for serine. The anticodon is underlined and the base pairing is shown with connecting lines.

Interestingly the gene order of 12S-Nad1 has been shown in two other pectinids,

Pecten maximus (Sellos et al . 1997 in (Sato and Nagashima 2001)) and Argopecten 46 irradians (Petten and Snyder 2007). In both these species there are tRNAs between the two genes. This gene order suggests that there is at least a partial conservation of gene order in the pectinids although gene order has been documented as not highly conserved in molluscs (Boore 1999), this gene order might indicate at least a partial conservation of gene order in the pectinids.

Chapter 3: Phylogenetic Analysis

1. Introduction

Chlamys islandica is part of the phylum Mollusca, class Bivalvia and family

Pectinidae. The phylum Mollusca is the second largest metazoan phylum after the

Arthropoda. They now inhabit marine, freshwater and terrestrial environments. They are believed to have evolved from a simple common ancestor which gives them the diversity they now posses (Doyle 1996). Molluscs exhibit a variety of appearances but share characters including a shell, a tissue layer called the mantle, a head and mouth at one end and anus at the other that discharges into the mantle cavity (Doyle 1996). In the modern mollusc these structures have been modified and some have been lost in some groups

(Doyle 1996). Mollusca is believed to be closely related to Platyhelminths, Nemertea, and Annelida. The exact phylogenetic relationship between the four phyla is not known, but since Platyhelminths are also simply organized and therefore molluscs are suspected to have arisen from a primitive acoelomate (Valentine 1973)

There are seven classes of molluscs but only six are extant. The class

Monoplacophora arose in the Cambrian and contains Neopilina and Vema . The class

Polyplacophora originated in the upper Cambrian and includes Chiton . The class 47

Scaphopoda originated in the Ordovician and includes Dentalium . The class Gastropoda originated in the Cambrian and includes slugs. The class Cephalopoda originated in the upper Cambrian and includes squids. The class Rostroconchia originated in the lower

Cambrian and went extinct in the Permian. The last class in the phylum Mollusca is

Bivalvia which are also known as Lamellibranchia or Pelecypoda. They originated in the

Cambrian and include the scallops such as Chlamys islandica (Doyle 1996) .

The ancestral mollusc is not known but a blueprint for it has been suggested as possessing all the characteristics of the soft body and a simple conical shell. The ancestral mollusc would have probably been a scavenger that would use the radula to scrape up food particles and had a simple respiratory system in which oxygenated water would have flowed over the gills. Based on this hypothetical ancestor, the evolution of the molluscs into different modes of life is possible. The bivalves would have adapted to a more sessile life, lost the radula and modified the gills for filter feeding (Doyle 1996).

The class Bivalvia contains molluscs that have a pair of calcareous valves that surround the soft part of the body (Clarkson 1998). They are bilaterally symmetrical with no head but a two-lobed mantel and shell (Hillmer and Lehmann 1980). Bivalves have been important in understanding the evolution processes in the geological records since they are readily preserved due to their shells. Some bivalves do exhibit a slow rate of evolution (Doyle 1996). The earliest known group of bivalves was the Fordilla which occurred in the Cambrian in North America, Scandinavia and Siberia (Clarkson 1998;

Doyle 1996), and Pojetaia from the Cambrian which was found in Australia (Doyle

1996). Though they started in the Cambrian, they became more diverse in the Ordovician

(Clarkson 1998; Doyle 1996). They underwent another adaptive radiation in the 48

Paleozoic when they displaced the brachiopods as the dominant suspension feeders in the shelf environment (Doyle 1996). They would inhabit niches close to shore for a while until the Permian extinction opened up the ocean to them to move to the offshore shelf

(Clarkson 1998). All of the subclasses of bivalves evolved in the Ordovician

(Palaeotaxodonta, Crytodonta, Pteriomorphia, Palaeoheterodonta, Heterodonta and

Anomalodesmata) (Clarkson 1998).

Chlamys islandica is part of the subclass Peteriomorphia. In the subclass

Peteriomorphia there are three orders, Arcoida, Mytiloida and Pterioida (which the scallops belong to) (Clarkson 1998). The genus Chlamys shows up in the fossil record in the Triassic (Hillmer and Lehmann 1980).

The scallops have been named using morphology in the past, but currently there is concern that convergent evolution gave rise to similar appearance and could account for some close groupings. This has lead to the suggestion that the systematics of the group should be revised (Shumway 1991).

A monophyletic group is a group of organisms that share a common ancestor in which all the descendants are included. If a phylogeny truly depicts the evolution of a group, then it will be monophyletic. A polyphyletic group is a group that contains some of the descendants of a common ancestor indicated for the group. A paraphyletic group contains most of the descendants but not the common ancestor indicated for the group. If a phylogeny shows either polyphyly or paraphyly, it is assumed that the systematics in use do not reflect the true evolutionary history of the group (Freeman and Herron 2004).

The phylogenetic relationship of Chlamys islandica to other scallops will be examined using the LSU and SSU rRNA sequences. The protein coding genes COI a and 49 b, CytB, Nad1, Nad3, Nad4 and Atp6 will be used to determine the phylogenetic relationships of the scallops among the bivalves. Implications of the findings are discussed.

2. Material and Methods

2.1 Sequence analysis

The sequences were analyzed in Bioedit 7.0 (Hall 1999). Two sequences were obtained for each gene, one from Chlamys A and one from Chlamys B (these correspond to two DNA samples sent for sequencing). Each sample was edited individually before being combined to obtain the final sequence. The forward and reverse sequences for each sample were edited by examining the electropherogram and a consensus sequence was created. The sequences obtained for samples A and B were then aligned and a consensus sequence was created to give the final sequence. Since there is not much variation in the mitochondrial genes and both samples were from Chlamys islandica, they should theoretically be the same sequence . If both agreed this gave more reliability to the sequence obtained. The end portions that were not reliable on the electropherograms (i.e. were not clear peaks) were then removed to obtain the final sequence for each gene (i.e. the edited sequences in Chapter 1). These sequences were used to create phylogenies with the exception of 16S. The 16S sequenced was not sequenced using universal primers since the universal primers did not amplify the 16S gene. Therefore a different region was amplified which does not correspond to the 16S sequences available for the scallops on GenBank. For this reason the 16S rRNA sequence used in the phylogenies was obtained from GenBank.

2.2 Phylogenies

2.2.1 Obtaining the sequences

The sequences for other bivalves (mainly scallops) used to create the phylogenies were obtained through GenBank. The references and accession numbers for those used in the 12S and 16S rRNA trees are shown in Table 5. The references and accession numbers for those used in the protein coding genes are in Table 6. The sequences were imported to Bioedit 7.0 (Hall 1999).

2.2.2 Creating the phylogenies

The sequences were imported to Mega 3.1 (Kumar et al . 2004). The Clustal W program within Mega 3.1 (Kumar et al . 2004) was used to align the sequences. The sequences were then made to be the same length. The aligned sequences were used to create minimum evolution trees by Mega 3.1 (Kumar et al . 2004). For the rRNA trees

Kimura-2 parameter was used for a nucleotide substitution and p-distance was used for the protein coding genes. Each tree was performed using 1000 bootstrap replicates to test for support for each branch.

Table 5. The GenBank accession numbers and references for the 39 scallops. These were used to create the 12S phylogeny, the 16S phylogeny and the combination of the 12S and 16S phylogeny and the outgroup of donkey’s foot ( Spondylus gaederopus ). GenBank Accession Species Gene number Citation Adamussium colbecki 16S AJ243882 (Canapa et al. 2000) 12S AJ571589 (Barucca et al. 2004) Aequipecten opercularis 16S AJ245397 (Canapa et al. 2000) 12S AJ571591 (Barucca et al. 2004) Amusium pleurnectes 16S DQ640830- (Barucca et al. 2004; Liu et al. 2001; DQ640845, Mahidol et al. 2007; Nguyen et al. DQ873917- 2006) DQ873919, AJ571616, AF362387 12S AJ571592 (Barucca et al. 2004) Annachlamys 16S DQ873924, (Nguyen et al. 2006) macassarensis DQ873923, DQ640847, DQ640846 12S - - Argopecten irradians 16S EU023915 (Petten and Snyder 2007) 12S Argopecten purpuratus 16S AJ972427, (Saavedra and Peña 2006) AJ972426 12S AM039763, AM039762 Argopecten ventricosus 16S AJ972430- (Saavedra and Peña 2006) AJ972428 12S AM039766- AM039764 Chlamys ferrari 16S AF362385 (Liu et al. 2001) 12S - - Chlamys glabra 16S AJ243574 (Canapa et al . 2000) 12S AJ571590 (Barucca et al. 2004) Chlamys islandica 16S AJ243573 (Canapa et al. 2000) 12S EU127905 Figure 9 Chlamys multistriata 16S AJ571617 (Barucca et al. 2004) 12S AJ571604 Chlamys varia 16S AJ586476, (Gonzalez-Tizon et al. 2003) AY650081, AJ243575 12S AJ571593 (Barucca et al . 2004) Comptopallium radula 16S DQ640889- (Na-Nakorn et al. 2006a) DQ640887 12S - - 52

Coralichlamys 16S AJ571608 (Barucca et al. 2004) madreporarum 12S AJ571598 Crassodoma gigantean 16S AJ972438, (Saavedra and Peña 2006) AJ972437 12S AM039774, AM039775 Decatopecten pilca 16S DQ873922, (Liu et al. 2001; Na-Nakorn et al . DQ873921, 2006a; Nguyen et al. 2006) DQ640890, AF362388 12S - - Decatopecten radula 16S DQ873920 (Nguyen et al. 2006) 12S - - Delectopecten vitreus 16S AJ571618 (Barucca et al . 2004) 12S - - Euvola vogdesi 16S AJ972432, (Saavedra and Peña 2006) AJ972431 12S AM039768, AM039767 Euvola ziczac 16S AJ972434, (Saavedra and Peña 2006) AJ972433 12S AM039770, AM039769 Gloripallium pallium 16S AJ571609 (Barucca et al. 2004) 12S AJ571599 Laevichlamys cuneata 16S AJ571610 (Barucca et al. 2004) 12S AJ571594 Laevichlamys 16S AJ571611 (Barucca et al. 2004) wilhelminae 12S AJ571595 Lyropecten nodosus 16S AJ972440, (Saavedra and Peña 2006) AJ972439 12S AM039776, AM039775 Mimachlamys nobilis 16S DQ873926- (Barucca et al. 2004; Na-Nakorn et DQ873934, al. 2006b) DQ640848- DQ640865, DQ640865, AJ571620 12S AJ571606 (Barucca et al. 2004)

Mimachlamys senatoria 16S DQ873935- (Na-Nakorn et al . 2006b; Nguyen et DQ873939, al . 2006) DQ640868- DQ640880, DQ640867, DQ640866 12S - - Minnivolva pyxidatus 16S DQ873942, (Na-Nakorn et al. 2006a; Nguyen et DQ640892, al. 2006) DQ640891 12S - - Mirapecten mirificus 16S AJ571612 (Barucca et al. 2004) 12S AJ571600 Mirapecten rastellum 16S AJ571613 (Barucca et al . 2004) 12S AJ571601 Mizuhopecten yessenosis 16S NC009081 (Sato and Nagashima 2001) 12S Nodipecten subnodosus 16S AJ972442, (Saavedra and Peña 2006) AJ972441 12S - - Pecten jacobaeus 16S AJ245394 (Canapa et al. 2000) 12S AJ571596 (Barucca et al . 2004) Pecten maximus 16S AJ972436, (Barucca et al . 2004; Liu et al . 2001; AJ972435, Saavedra and Pena 2005; Saavedra AY650056- and Peña 2006) AY650084, AJ571619, AF362339 12S X95497, (Barucca et al. 2004; Berschick 1997; AM039772, Saavedra and Peña 2006) AM039771, AJ571597 Pecten novaezelandiae 16S AJ972446, (Saavedra and Peña 2006) AJ972445, AY650055 12S AM039782, (Saavedra and Peña 2006) AM039781 Placopecten 16S DQ088274 (Smith and Snyder 2007) magellanicus 12S Semipallium amicum 16S AJ571614 (Barucca et al. 2004) 12S AJ571602 Semipallium dringi 16S AJ571615 (Barucca et al. 2004) 12S AJ571603

Semipallium folvicostata 16S DQ640896, (Na-Nakorn et al. 2006a; Nguyen et DQ873925, al . 2006) DQ640895 12S - - Zygochlamys patagonica 16S AJ972448, (Saavedra and Peña 2006) AJ972447 12S AM039784, AM039783 Spondylus gaederopus 16S AJ571621 (Barucca et al. 2004) 12S AJ571607

Table 6. The GenBank accession numbers, classification and references for 13 bivalves with complete mitochondrial genomes sequenced and a Polyplacophora that is used as an outgroup for the phylogenies. The sequences were used for the COI, Nad1, CytB, Nad3, Nad4 and ATP6 phylogenies. GenBank Accession Class, Species number Family Citation Acanthocardia NC008452 Bivalvia, (Dreyer and Steiner 2006) tuberculata Cardiidae Argopecten irradians NC009687 Bivalvia, (Petten and Snyder 2007) Pectinidae Chlamys islandica Appendix 2 Bivalvia, Figures 11-17 in Chapter 1. Pectinidae Crassostrea gigas NC001276 Bivalvia, (Kim et al. 1999) Ostreidae Crassostrea virginica NC007175 Bivalvia, (Milbury and Gaffney 2005) Ostreidae Hiatella artica NC008451 Bivalvia, (Dreyer and Steiner 2006) Hiatellidae Lampsilis ornata NC005335 Bivalvia, (Serb and Lydeard 2003) Lampsilinae Mizuhopecten NC009081 Bivalvia, (Sato and Nagashima 2001) yessoensis Pectinidae Mytilus edulis NC006161 Bivalvia, (Boore et al . 2004; Hoffmann Mytilinae et al. 1992) Mytilus DQ399833 Bivalvia, (Venetis et al. 2007) galloprovinvialis Mytilinae

Mytilus trossulus DQ198225 Bivalvia, (Burzynski et al. 2006; Mytilinae Zbawicka et al . 2007) Placopecten NC007234 Bivalvia, (Smith and Snyder 2007) magellanicus Pectinidae Vererupis AB065375, Bivalvia, (Okazaki and Ueshima 2001) philippinarium AB065374 Veneridae Katharina tunicata NC001636 Polyplacophora, (Boore and Brown 1994) Mopaliidae

3. Results and Discussion

The LSU rRNAs (16S) for the scallops were used to determine the phylogenetic relationships shown in Figure 28. There are 39 species of scallops and donkey’s foot,

Spondylus gaederopus, which is another bivalve that also belongs to the subclass 56

Pteriomorphia and the family Spondylidae, which is used as the outgroup. Scallops belong to the family Pectinidae, but down to the family level Spondylus gaederopus shares the same taxonomy which makes it a closely related species and a good outgroup.

In the phylogeny in Figure 28 Chlamys islandica groups with Mizuhopecten yessenosis with a bootstrap valve of 58 which is weak support for this clade, but the clade for Mizuhopecten yessenosis, Chlamys islandica and Crassodoma gigantea has a bootstrap value of 94 which gives strong support to the clade. The living relatives of

Chlamys islandica are found in the north Pacific (Shumway 1991). Mizuhopecten yessenosis is found in the north Pacific around Japan and China, as well as in West

Russia, Europe and Canada (Minchin 2003). Therefore it is likely that Mizuhopecten yessenosis could be closely related to Chlamys islandica as the phylogeny depicts.

Chlamys islandica does not group with any of the other four Chlamys species in the phylogeny. The only two Chlamys that group together are Chlamys multistrata and

Chlamys varia . This indicates that the genus Chlamys is not a monophyletic group but polyphyletic. This has also been demonstrated by Barucca et al . (2004). As mentioned above, when many of the scallops were being named they were named by morphology which may not recover their true evolutionary relationships (Shumway 1991). Since

Linnaeus’s Systema Naturae was published in 1758 almost 7000 species and subspecies of the family Pectinidae have been described from both living species and fossil records.

Approximately 80% of these were placed into only five genera: Pecten, Chlamys,

Hinnities, Amusium and Aequipecten (Shumway 1991). Now many use generic names because the scientific names have been classified by shell outlines that evolved repetitively during their evolutionary history due to modes of life (Shumway 1991). This 57 is why some are known by more than one genus such as Mizuhopecten which is also known as Patinopecten (Sato and Nagashima 2001). Though some genera show monophyly such as Pecten, Argopecten and Euvola, many do not.

Other genera in Figure 28, such as Mirapecten and Mimachlamys, are also polyphyletic genera. Also, other genera are paraphyletic such as: Semipallium,

Laevichlamys, and Decatopecten. For other genera there is only one representative in the phylogeny such as Placopecten, Aequipecten, Delectopecten, Crassodoma, Zygochlamys,

Minnivola, Coralichlamys, Adamussium, Comptopallium, Gloripallium, Annachlamys,

Lyropecten, Nodipecten and Amusium. Since there is only one representative of these genera, whether they are monophyletic or not cannot be determined. There needs to be at least two or more to determine monophyly, but as always the more species present in a genus the more support there is for the grouping. Therefore even the three genera that seem to be monophyletic ( Pecten, Argopecten and Euvola ) could deviate if more members are used. Since they are the only ones available then, based on these species the genera are monophyletic.

The phylogenetic relationship of 30 scallops are examined using the SSU rRNA

(12S) with Spondylus gaederopus used as an outgroup (Figure 29). Chlamys islandica again groups with Mizuhopecten yessenosis with a bootstrap value of 81 which gives good support to this clade. Also Crassodoma gigantea also groups with Chlamys islandica and Mizuhopecten yessenosis with a bootstrap value of 99 which is very strong support. The 12S phylogeny agrees with the 16S tree for the position of Chlamys islandica with Mizuhopecten yessenosis but has a better support for this clade. Many of the clades are the same in the 12S as with the 16S. There are a few differences such as 58

Placopecten magellanicus groups with Zygochlamys patagonica instead of Mimachlamys nobilis. The grouping of Placopecten magellanicus with Zygochalmys patagonica has stronger support (51 compared to 30) than the grouping of Placopecten magellanicus.

Neither of these values give strong support. Mimachlamys nobilis is closer to Chlamys islandica, Mizuhopecten yessenosis and Crassadoma gigantea clade than in the 16S tree.

Adamussium colbecki is closer to Placopecten magellanicus and Zygochlamys patagonica than the Argopecten genus. There is a higher bootstrap value in this tree (59 compared to 36) for the grouping of Adamussium colbecki. Neither of these are strongly supported. The 12S tree also shows that Chlamys is not a monophyletic genus. In the

12S tree the genus Semipallium is also monoplyletic as well as Pecten, Argopecten and

Euvola . In the 16S tree Semipallium folvicostata does not fit with the other members of the genus, so the clade is paraphyletic based on the data. Since no 12S sequence is available for Semipallium folvicostata, the same problem is not observed in the tree.

Therefore based on the 16S rRNA tree, Semipallium is paraphyletic.

The phylogenetic relationship between the scallops and Spondylus gaederopus as the outgroup using the combination of the 16S and 12S data is shown in Figure 30.

Since there was a lot of similarity between the 16S and 12S trees in Figures 28 and 29, many of the groups are the same. Again Mizuhopecten yessenosis groups with Chlamys islandica with a bootstrap valve of 58 which is weak support, but the 12S tree provided good support for this grouping. Crassodoma gigantea also groups with these two with a bootstrap value of 94 which is strong support. Semipallium is not monophyletic but

Pecten, Argopecten and Euvola still maintain monophyly. Chlamys does not show 59 monophyly and therefore is not a monophyletic group based on these phylogenies. The groupings are similar to those of the 16S phylogeny.

Since many of these genera do not show monophyly, this demonstrates that the systematics of the family Pectinidae does need to be revised as Shumway (1991) has stated. The molecular data presented here supports previous data sets (Barucca et al .

2004; Canapa et al . 1998; Liu et al . 2007; Lydeard et al . 2000; Saavedra and Peña 2006), in that the most genera within the family Pectinidae are not monophyletic, but either polyphyletic or paraphyletic. Therefore this supports the hypothesis that many shared morphological characteristics are due to convergent evolution and not true evolutionary relationships. 60

58 Chlamys islandica 94 Mizuhopecten yessoensis 52 Crassodoma gigantea 9 Chlamys ferrari 17 Mimachlamys senatoria Chlmays multistriata 20 74 Chlamys varia

19 Zygochlamys patagonica Minnivolva pyxidatus

98 Semipallium amicum 27 Semipallium dringi

38 95 Semipallium folvicostata Laevichlamys cuneata 96 48 Laevichlamys wilhelminae 95 Coralichlamys madreporarum 36 Placopecten magellanicus 30 Mimachlamys nobilis Adamussium colbecki Argopecten irradians 36 99 Argopecten purpuratus 64 Argopecten ventricosus 79 100 Comptopallium radula 84 Decatopecten radula

42 Decatopecten pilca Mirapecten rastellum 30 99 Gloripallium pallium Mirapecten mirificus 30 Annachlamys macassarensis 20 100 Lyropecten nodosus 90 Nodipecten subnodosus Amusium pleuronectes 71 91 Euvola vogdesi 80 Euvola ziczac

55 Pecten maximus Pecten jacobaeus 100 Pecten novaezelandiae Chlamys glabra 99 Aequipecten opercularis Delectopecten vitreus Spondylus gaederopus

0.05 Figure 28. Phylogenic relationship of 39 scallop species and Spondylus gaederopus as an outgroup using the partial 16S rRNA sequence. Minimum evolution analysis, substitution model Kimura 2- parameter and 1000 bootstrap replicates were used. The scale represents 0.05 substitutions per site. 61

83 Laevichlamys wilhelminae 98 Corilachlamys madreporarum

98 Laevichlamys cuneata Semipallium dringi 18 68 Semipallium amicum Adamussium colbecki

59 Placopecten magellanicus 9 51 Zygochlamys patagonica

81 Chlamys islandica 99 Mizuhopecten yessoensis Crassadoma gigantea 54 Mimachlmays nobilis

23 Chlamys multistriata 34 90 Chlamys varia

93 Argopecten ventricosus 99 Argopecten purpuratus

34 Argopecten irradians Nodipecten subnodosus 100 Lyropecten nodosus

47 59 97 Euvola ziczac Euvola vogdesi

93 Amusium pleuronectes Pecten novaezelandiae 58 97 Pecten maximus 88 Pecten jacobaeus Mirapecten mirificus

97 Mirapecten rastellum 97 Gloripallium pallium Aequipecten opercularis 98 Chlamys glabra Spondylus gaederopus

0.05 Figure 29. Phylogenic relationship of 30 scallop species and Spondylus gaederopus as an outgroup using the partial 12S rRNA sequence. Minimum evolution analysis, substitution model Kimura 2- parameter and 1000 bootstrap replicates were used. The scale represents 0.05 substitutions per site. 62

58 Chlamys islandica 94 Mizuhopecten yessoensis 52 Crassodoma gigantea 9 Chlamys ferrari 18 Mimachlamys senatoria Chlmays multistriata 20 74 Chlamys varia

19 Zygochlamys patagonica Minnivolva pyxidatus

98 Semipallium amicum 27 Semipallium dringi

39 95 Semipallium folvicostata Laevichlamys cuneata 96 48 Laevichlamys wilhelminae 95 Coralichlamys madreporarum 36 Placopecten magellanicus 30 Mimachlamys nobilis Adamussium colbecki Argopecten irrandians 36 99 Argopecten purpuratus 64 Argopecten ventricosus 79 100 Comptopallium radula 84 Decatopecten radula

55 Pecten maximus Pecten jacobaeus 100 Pecten novaezelandiae Chlamys glabra 99 Aequipecten opercularis Delectopecten vitreus Spondylus gaederopus

0.05 Figure 30. Phylogenic relationship of 39 scallop species and Spondylus gaederopus as an outgroup using the partial 12S rRNA and 16S rRNA sequences. Minimum evolution analysis, substitution model Kimura 2-parameter and 1000 bootstrap replicates were used. Scale represents 0.05 substitutions per site. 63

The phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata

(black chition), from the class Polyplacophora, for the protein coding genes COIb, Nad1,

CytB, Nad3, and Nad4 are depicted in Figures 31, 33, 34, 35 and 36 respectively. In these phylogenies the grouping within the family Pectinidae varies but the monophyly of the family Pectinidae is constant with strong support in all with bootstrap values of 100 and 99. Others have demonstrated monophyly of the family Pectinidae, but only had a small sample group of two (Passamaneck et al . 2004). Here there are only four members of this family ( Chlamys islandica, Mizuhopecten yessenosis, Argopecten irradians and

Placopecten magellanicus ) which is not a good representation since there are over 7000 species and subspecies in the family Pectinidae (Shumway 1991). The small representation presented here is due to lack of completed Pectinidae mitochondrial genomes. Since the protein coding genes are not as commonly sequenced as the rRNA genes the best source for these genes are completed mitochondrial genomes. Though this is a small sample size, these four members do demonstrate a monophyletic group for the family Pectinidae. Each gene evolves individually as an individual unit. Therefore each gene phylogeny demonstrates a distinct evolutionary relationship. Since there are five genes that all give strong support to the monphyly of the family Pectinidae, using these four members, the family Pectinidae is monophyletic.

In Figures 31 and 33-36 the families Mytilinae and Ostreidae are also monophyletic families. There are three representatives of the family Mytilinae: Mytilus edulis, Mytilus galloprovinciales and Mytilus trossulus . There are two representatives of the family Ostreidae: Crassostrea gigas and Crassostrea virginica . As with the

Pectinidaes, these are small sample groups due to lack of completed mitochondrial 64 genomes. The monphyly of these families are strongly supported with bootstrap values of 100 in all phylogenies for the family Mytilinae and ranging from 94 to 100 for the family Ostreidae. The other four families in the class Bivalvia present in the phylogenies

(Cardiidae, Hiatellidae, Lampsilinae and Veneridae) only have one member present and therefore not enough data to observe groups of monophyletic, polyphyletic or paraphyletic status.

The genus Mytilus (family Mytilinae) and the genus Chlamys (family Pectinidae) become present in the fossil record in the Triassic (Hillmer and Lehmann 1980). These are not necessarily the earliest members in their respective families. When comparing branch length (Figure 31 and 33-36) the family Pectinidae has an earlier divergence compared to the family Mytilinae. This could be biased based on only one genus present in the family Mytilinae compared to four genera for the family Pectinidae. The fossil record dates for the other genera present in the family Pectinidae could not be found.

These branch lengths indicate that the family Mytilinae had a long period of stasis followed by a more recent period of divergence. The divergence of the family Pectinidae occurred before the family Mytilinae divergence. The difference in the branch lengths could also be due to different rates of evolution. This would indicate that the family

Pectinidae has a faster rate of evolution than the family Mytilinae. Since these dates are for the genera Mytilus and Chlamys the branch lengths could also be misleading since three species in the genus Mytilus are present and only one species is present for the genus Chlamys. The branch observed for Chlamys is the divergence from another genus

(Mizuhopecten ) and the branches among the family Mytilinae are among the genus

Mytilus. It would be expected that the branches among a genus are shorter than within a 65 family. Since there is a lack of data no valid conclusion could be made on the rate of divergence or rate of evolution.

80 Argopecten irradians

100 Placopecten magellanicus Mizuhopecten yessoensis 37 100 Chlamys islandica Mytilus galloprovincialis

50 100 Mytilus trossulus 98 Mytilus edulis 74 Crassostrea virginica

100 29 Crassostrea gigas Lampsilis ornata 69 Hiatella arctica Acanthocardia tuberculata Venerupis philippinarum Katharina tunicata

0.05

Figure 31. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata , using the partial sequence of the COI gene (b). A minimum evolution tree was produced using p-distance nucleotide substitution and 1000 bootstrap replicates. The scale represents 0.05 substitutions per site.

78 Placopecten magellanicus 99 Mizuhopecten yessoensis

49 Argopecten irradians Hiatella arctica

31 77 Acanthocardia tuberculata 54 Venerupis philippinarum

100 Crassostrea virginica 100 Crassostrea gigas Mytilus galloprovincialis

100 Mytilus trossulus 93 Mytilus edulis Chlamys islandica Lampsilis ornata

0.05 Figure 32. Phylogenetic relationship of 13 bivalves with no outgroup using the partial sequence of the COI gene (a) suspected of being a pseudogene. A minimum evolution tree was produced using a p-distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.05 substitutions per site.

Figure 31 depicts the phylogenetic relationship of 13 bivalves using the gene

COIb gene and Katharina tunicata (black chition), from the class Polyplacophora, as an outgroup. Figure 32 depicts the phylogenetic relationship of 13 bivalves using the gene

COIa gene with no outgroup. As discussed in Chapter 1, the COIa gene is a pseudogene

(i.e. a Numt). When the two phylogenies are examined, the position of Chlamys islandica do not agree. In the COIb phylogeny Chlamys islandica groups with

Mizuhopecten yessenosis with a bootstrap value of 100 which gives strong support for the clade, and Argopecten irradians and Placopecten magellanicus group with a bootstrap value of 80 which gives good support for the clade. The family Pectinidae ( Chlamys islandica, Mizuhopecten yessenosis, Argopecten irradians and Placopecten magellanicus ) group together with a bootstrap value of 100 which gives strong support 67 for the monophyly of the family Pectinidae. In the COIa phylogeny Chlamys islandica does not group with the Pectinidaes, but seems to be closer to Lampsilis ornata. The other members of the family Pectinidae form a clade with a bootstrap value of 98.

Therefore COIa does not support monophyly, but since it is a pseudogene it does not give the true evolutionary relationship (Bensasson et al . 2001; Lopez et al . 1994). Since COIa does not depict the true evolutionary relationship the polyphyly presented in this phylogeny does not hold any significance. The monophyly in the COIb gene does have significance and is strongly supported by the bootstrap value.

The divergence depicted in Figure 32 for the COIa gene would be due to faster rate of mutation (Adams and Palmer 2003; Bensasson et al . 2001). Once it transferred to the nuclear genome the protein either could not import back into the mitochondria due to high hydrophobicity or the protein would have been changed or premature stop codons inserted because of the difference of the invertebrate mitochondrial genetic code and the standard genetic code (Adams and Palmer 2003). Since it is non-functional there would not be selective pressure on the gene to preserve function, and it would mutate faster than a functional gene. Since it was amplified by the universal primers for COI it can be assumed that the recognition site of the primers has not mutated and therefore it out competed the mitochondrial copy of COI in the PCR (Bensasson et al . 2001).

53 Mizuhopecten yessoensis 74 Chlamys islandica 100 Placopecten magellanicus

72 Argopecten irradians Mytilus galloprincialis

50 100 Mytilus trossulus 79 Mytilus edulis Hiatella arctica

62 Acanthocardia tuberculata 52 Venerupis philippinarum Lampsilis ornata

72 Crassostrea virginica 99 Crassostrea gigas Katharina tunicata

0.05 Figure 33. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad1 gene. A minimum evolution tree was produced using a p-distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.05 substitutions per site.

Figure 33 depicts the phylogenetic relationship of 13 bivalves and the outgroup

Katharina tunicata using the Nad1 gene. Chlamys islandica and Mizuhopecten yessenosis group together with a bootstrap value of 53 which shows weak support for this clade. The clade that includes Chlamys islandica, Mizuhopecten yessenosis and

Placopecten magellanicus groups together with a bootstrap value of 74 which is better support than the clade of Mizuhopecten yessenosis and Chlamys islandica. The family

Pectinidae shows monophyly with a bootstrap value of 100 which is strong support.

61 Mizuhopecten yessoensis 60 Chlamys islandica 100 Argopecten irradians

25 Placopecten magellanicus Hiatella arctica Mytilus galloprovincialis 26 44 100 Mytilus trossulus 59 Mytilus edulis 59 Acanthocardia tuberculata 87 Venerupis philippinarum Crassostrea virginica 100 Crassostrea gigas Lampsilis ornata Katharina tunicata

0.05 Figure 34. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Cytb gene. A minimum evolution tree was produced using a p-distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.05 substitutions per site.

Figure 34 depicts the phylogenetic relationship of 13 bivalves and the outgroup

Katharina tunicata using the CytB gene. Chlamys islandica and Mizuhopecten yessenosis group together with a bootstrap value of 61 which is weak support for this clade. The clade of Mizuhopecten yessenosis, Chlamys islandica and Argopecten irradians groups together with a bootstrap value of 60 which is weak support. The family Pectinidae demonstrated monophyly with a bootstrap value of 100 which is strong support. 70

75 Argopecten irradians 69 Placopecten magellanicus 99 Mizuhopecten yessoensis

38 Chlamys islandica Lampsilis ornata

45 87 Crassostrea virginica 94 Crassostrea gigas Acanthocardia tuberculata

17 Hiatella arctica 35 Venerupis philippinarum Mytilus galloprovinvialis

100 Mytilus trossulus 99 Mytilus edulis Katharina tunicata

0.1

Figure 35. Phylogenetic relationships of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad3 gene. A minimum evolution tree was produced using p-distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.1 substitutions per site.

Figure 35 depicts the phylogenetic relationship of 13 bivalves and the outgroup

Katharina tunicata using the Nad3 gene. This is the only true phylogeny that does not group Mizuhopecten yessenosis and Chlamys islandica together. Argopecten irradians and Placopecten magellanicus group together with a bootstrap value of 75 which give support to the clade. The clade of Argopecten irradians, Placopecten magellanicus and

Mizuhopecten yessenosis has a bootstrap value of 69 which gives weak support. The family Pectinidae demonstrates monophyly with a bootstrap value of 99 which is strong support. 71

100 Mizuhopecten yessoensis 42 Chlamys islandica 100 Placopecten magellanicus 81 Argopecten irradians Crassostrea virginica 49 100 Crassostrea gigas Hiatella arctica

100 87 Acanthocardia tuberculata 75 Venerupis philippinarum Mytilus galloprovincialis

100 Mytilus trossulus 99 Mytilus edulis Lampsilis ornata Katharina tunicata

0.05

Figure 36. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Nad4 gene. A minimum evolution tree was produced using p-distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.05 substitutions per site.

The phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the Nad4 gene is depicted in Figure 36. Chlamys islandica and Mizuhopecten yessenosis group together with a bootstrap value of 100 which is strong support for this clade. The clade of Chlamys islandica, Mizuhopecten yessenosis and Placopecten magellanicus has a bootstrap value of 42 which is weak support for this clade. The family Pectinidae forms a clade with a bootstrap value of 100 which is strong support for the monophyletic relationship.

95 Chlamys islandica

58 Lampsilis ornata Crassostrea virginica 50 100 Crassostrea gigas

43 Acanthocardia tuberculata 45 Hiatella arctica 90 Venerupis philippinarum Argopecten irradians

99 Placopecten magellanicus 86 Mizuhopecten yessoensis Mytilus galloprovincialis

100 Mytilus trossulus 99 Mytilus edulis Katharina tunicata

0.05 Figure 37. Phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the partial sequence of the Atp6 gene sequence. A minimum evolution tree was produced using p- distance nucleotide substitution and 1000 bootstrap replicates. Scale represents 0.05 substitutions per site.

The phylogenetic relationship of 13 bivalves and the outgroup Katharina tunicata using the Atp6 gene is depicted in Figure 37. Chlamys islandica does not group with the other pectinids in this phylogeny. Otherwise the family Pectinidae is monophyletic with a 99 bootstrap value. As discussed in Chapter 1, the Atp6 sequence obtained is thought to be a pseudogene because the amino acid sequence contains stop codons (Figure 24).

This phylogeny does not depict the true grouping of Chlamys islandica since a pseudogene can have more mutations than a functional gene (Adams and Palmer 2003;

Bensasson et al . 2001; Lopez et al . 1994). Therefore there is no weight placed on this phylogeny. The phylogenies for the other protein coding genes all agree in the monophyly of the family Pectinidae and they were created using the actual genes and therefore more weight would be placed on them.

Chapter 4. Conclusions

Prior to this study three genes were available on GenBank for the mitochondrial

DNA of Chlamys islandica , the 12S, 16S and COI genes. These genes have amounted to a total of 1921bp for the mitochondrial DNA on GenBank. This study had added a different region to the 16S gene and Nad1, Nad3, Nad4 and CytB to the list of partial genes sequenced as well as the intergenic region between 12S and Nad1. In total this study has produced 2211bp (this total does not include the pseudogenes) in which 1565bp are different than those present on GenBank. This work now brings the total of known bases for the mitochondrial DNA to 3486bp (includes those previously on GenBank and those presented here). The percentage of the genome sequenced in this study is 9.2% and the total known now is 14.5%. These percentages are based on 24kb (average of 23kb and 25kb). The portion of the genome sequenced has laid the ground work for the completed genome. Prior to this study we lacked any gene order in the mitochondrial genome of Chlamys islandica . This study has provided some clarity to the possible gene order for two genes as well as a possible tRNA.

This study has contributed both to a better understanding of the problems of taxonomy within the genus Chlamys and to a better understanding of the relationship of

Chlamys islandica to other members of the pectinid clade, as well as helping to increase the existing data base for bivalve mitochondrial DNA. My results suggest a close taxonomic association between Mizuhopecten yessenosis and Chlamys islandica, a result that is sensible given the knowledge that the closest relative of Chlamys islandica is believed to be in the north pacific (Shumway 1991) which is where Mizuhopecten yessenosis is found (Minchin 2003). In addition, because the results strongly support the 74 assertion that the genus Chlamys is not monophyletic, the need for revision of the taxonomy of the family Pectinidae in order to depict the true evolutionary history is strongly supported.

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Appendix 1

Table 7. All primers used in the study, their target and primer sequence Primer Target Primer Sequence (5 ’→3’) LCO1490 COI GGTCAACAAATCATAAAGATATTGG Hco2198 COI TAAACTTCAGGGTGACCAAAAAATCA COXF COI GTTYTWATTGGDGGGTTTGG* COXR COI TGMCCCCAYACAATAAAVCC* 16SL 16S TCGTACCTTTTGCATCATCATGG 16SR 16S TTTTGCCGAGTTCCTTTAGC 12S-F 12S AGACATGGATTAGATACCC 12S-R 12S ACCCCTACCTTGTTACGACTT ATP6L Atp6 TTRAGGACTTAYTTTGATCAGTTG* ATP6H Atp6 GCCAAAAKAATATGACCTCAM* cobF424 CytB GGWTAYGTWYTWCCWTGRGGWCARAT* cobR876 CytB GCRTAWGCRAAWARRAARTAYCAYTCWGG* ND1F Nad1 TTTGAGCGRAARGTKTTRSC* ND1R Nad1 CATAWARRCCSCCCCAAAAA* ND3F Nad3 GGKTBMGGGATTCYCARAAG* ND3R Nad3 AAWGAYTCHACYTCAAABACCA* ND4F Nad4 GTTTTGGYTGGGVGCTYTWAT* ND4R Nad4 CTCAACRTGMGCYTTAGTYAACC* 12S-ND1L 12S-Nad1 ACTTAGGAGTAAGGGGAGATTAATATGCTT 12S-ND1R 12S-Nad1 ATAAAACAGGGACCTAGAAAAAACAACAC *R=A or G; Y=C or T; W=A or T; K=G or T; M=A or C; S=G or C; B=G, T or C; H= A, T or C; V=G, A or C; D=G, A or T

Appendix 2

Table 8. The GenBank accession numbers for genes sequenced in this study. Gene GenBank Accesion # COI a EU127910 COIb EU564118* CytB EU127908 Nad1 EU127909 Nad3 EU564116* Nad4 EU564118* 12S EU127905 16S EU127906 Atp6 EU127907 *Submitted to GenBank but not yet on the server