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University of Iowa Iowa Research Online Theses and Dissertations Spring 2015 Evolution of meiosis genes in sexual vs. asexual Potamopyrgus antipodarum Christopher Steven Rice University of Iowa Copyright 2015 Christopher Steven Rice This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/1739 Recommended Citation Rice, Christopher Steven. "Evolution of meiosis genes in sexual vs. asexual Potamopyrgus antipodarum." MS (Master of Science) thesis, University of Iowa, 2015. http://ir.uiowa.edu/etd/1739. Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Biology Commons EVOLUTION OF MEIOSIS GENES IN SEXUAL VS. ASEXUAL POTAMOPYRGUS ANTIPODARUM by Christopher Steven Rice A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Biology in the Graduate College of The University of Iowa May 2015 Thesis Supervisors: Associate Professor John M. Logsdon, Jr. Associate Professor Maurine Neiman Copyright by CHRISTOPHER STEVEN RICE 2015 All Rights Reserved Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL MASTER'S THESIS This is to certify that the Master's thesis of Christopher Steven Rice has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Biology at the May 2015 graduation. Thesis Committee: John M. Logsdon Jr., Thesis Supervisor Maurine Neiman, Thesis Supervisor Bryant McAllister ACKNOWLEDGMENTS I would like to acknowledge Dr. Maurine Neiman and Dr. John Logsdon for their outstanding mentorship and guidance in the completion of this project. I would like to thank all members of the Neiman and Logsdon labs, for your incredibly helpful contributions and advice. I would like to thank Dr. Jeffrey Boore for his assistance in our bioinformatics endeavors, the University of Iowa Biology Department, and the National Science Foundation for their support. Lastly I'd like to thank my thesis committee for their insight and assistance in the completion of my research. ii ABSTRACT How asexual reproduction affects genome evolution, and how organisms that are ancestrally sexual alter their reproductive machinery upon becoming asexual are both central unanswered questions in evolutionary biology. While these questions have been addressed to some extent in organisms such as asexual clams, rotifers, ostracods, arthropods, and fungi, the most powerful and direct tests of how sex and its absence influence evolution requires direct comparisons between closely related and otherwise similar sexual and asexual taxa. Here, I quantify the rates and patterns of molecular evolution in the meiosis-specific genes Msh4, Msh5, and Spo11 in multiple sexual and asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail. Because asexual P. antipodarum reproduce apomictically (without recombination), genes used only for meiosis should be under relaxed selection relative to meiosis-specific genes in sexual P. antipodarum, allowing me to directly study how asexuality affects the evolution of meiosisspecific genes. Contrary to expectations under relaxed selection, I found no evidence that these meiosis-specific genes are degrading in asexual P. antipodarum; instead they display molecular patterns consistent with purifying selection. The presence of intact meiosis-specific genes in asexual P. antipodarum hints that the asexuals may maintain the ability to perform meiosis despite reproducing apomictically. Asexual meiotic capability suggests that some meiotic components may persist or acquire a new role in these asexuals. iii PUBLIC ABSTRACT How asexual reproduction affects genome evolution, and how ancestrally sexual organisms alter their reproductive machinery upon becoming asexual are central unanswered questions in evolutionary biology. While these questions have been addressed to some extent in different asexual species, the most powerful tests of how sex and its absence influence evolution requires direct comparisons between closely related and otherwise similar sexual and asexual taxa. I address these questions by studying the evolution of genes critical to sexual reproduction in sexual and asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail. Because genes used only for sexual reproduction should have little or no functional relevance for asexuals, I can use this approach to study the effects of asexuality on the evolution of genes required only for sexual reproduction, which has implications for genome evolution in the absence of sex. I discovered that genes necessary for sexual reproduction show no evidence for being lost in asexual P. antipodarum. These results suggest asexuality may result in the loss of some, but not all, components of sexual reproductive machinery or that not enough time has passed for meiosis genes to be lost. Comparing evolutionary patterns between sexual and asexual organisms will help the scientific community understand the benefits and limitations of reproducing sexually. Understanding the consequences of sexual reproduction will illuminate fundamental biological and human health questions, ranging from the importance of biological diversity to the source and transmission of disease to dynamics between humans (who reproduce sexually) and parasites (which are often asexual). iv TABLE OF CONTENTS List of Tables ............................................................................................................................... vi List of Figures ............................................................................................................................ vii Chapter 1: Introduction .................................................................................................................1 Determining Evolutionary Consequences of Asexuality for Meiosis-Specific Genes ..............1 Evolution of Genes under Relaxed Selection.............................................................................2 Meiosis in Sexual Reproduction ................................................................................................3 Asexual Reproduction ................................................................................................................5 Using the Meiosis Detection Toolkit to Detect Evidence of Relaxed Selection........................6 Study System: Potamopyrgus antipodarum ..............................................................................8 Summary ..................................................................................................................................10 Chapter 2: Materials and Methods ..............................................................................................11 Genome Sequencing and Assembly .........................................................................................11 Gene Assembly and Annotation ..............................................................................................11 PCR Amplification and Gel Electrophoresis of Meiotic Genes...............................................13 Cloning of PCR Products .........................................................................................................20 Screening of Clones .................................................................................................................20 Plasmid Extraction and Preparation .........................................................................................23 Processing and Analysis of Sanger Sequences ........................................................................23 Chapter 3: Results .......................................................................................................................26 Accumulated Mutations of Sexual and Asexual Potamopyrgus antipodarum ........................26 Evolutionary Rates of Sexual vs. Asexual Genes ....................................................................26 Chapter 4: Discussion..................................................................................................................35 Chapter 5: Summary....................................................................................................................38 References ...................................................................................................................................39 v LIST OF TABLES Table 1. Expected Sequence Patterns for Corresponding Modes of Selection. ..............................9 2. Msh4 PCR Primers. .........................................................................................................14 3. Msh5 PCR Primers. .........................................................................................................15 4. Spo11 PCR Primers.........................................................................................................16 5. Stock and Working Concentration of PCR Reagents. ....................................................17 6. Thermocycler Program used in PCR Reactions. .............................................................18 7. Stock and Working Concentrations of PCR Screening Reagents. ..................................21 8. Thermocycler Program used in PCR Screening Reactions. ............................................22 9. IUPAC Degenerate Codes at Heterozygous Sites for Maximum Likelihood Analysis. .25 10. Open Reading Frames in Sexual and Asexual P. antipodarum. .....................................27 vi LIST OF FIGURES Figure 1. Mitochondrial Phylogeny of Potamopyrgus antipodarum....................................... 12 2. Gene Models and PCR Strategy for Meiosis-Specific Genes. ................................. 19 3. Msh4 Polymorphism. ............................................................................................... 28 4. Msh5 Polymorphism. ............................................................................................... 29 5. Spo11 Polymorphism. .............................................................................................. 30 6. Maximum Likelihood Analysis of Msh4 ................................................................. 31 7. Maximum Likelihood Analysis of Msh5 ................................................................. 32 8. Maximum Likelihood Analysis of Spo11. ............................................................... 33 9. Evolutionary Rate of Sexual and Asexual Msh5 Coding Region. ........................... 34 vii Chapter 1: Introduction Determining Evolutionary Consequences of Asexuality for Meiosis-Specific Genes My thesis will address the consequences of asexuality for genome evolution by studying how meiosis genes critical to sexual reproduction evolve in asexual lineages. I have compared meiotic genes of multiple diverse obligate sexual and obligate asexual lineages of the snail Potamopyrgus antipodarum. My research aims to capture the early evolutionary genomic consequences of asexuality, which are largely unstudied in eukaryotes outside of arthropods, clams, rotifers, ostracods, and fungi (reviewed by Nomark 2003, Schurko et al. 2009, Schurko et al. 2010). By comparing young and relatively old asexual lineages I will provide novel observations of evolution within meiosis genes hypothesized to be under relaxed selection in asexuals. Comparing meiosis gene evolution between reproductive modes in P. antipodarum is a powerful strategy in this context because asexual organisms typically do not require meiosis to reproduce (Naumova et al. 2001, Hojsgaard et al.2013, Wang et al. 2014), meaning that genes only used for meiosis should experience relaxed selective constraint relative to their sexual counterparts. How relaxed selection affects molecular evolution in meiosis genes has yet to be addressed in systems that allow direct comparisons between sexuals and otherwise similar asexuals. P. antipodarum features closely related, coexisting, and ecologically similar sexual and asexual individuals, meaning I can directly compare the evolution of orthologs in sexually vs. asexually transmitted genomes. These comparisons will provide powerful tests of hypotheses for pseudogenization and decay of asexual meiosis genes. The expectations of meiosis gene decay in asexuals are complicated by the fact that some asexual lineages use alternate forms of meiosis, likely mediated by modification to subsets of proteins within otherwise maintained core meiotic machinery (Srinivasan et al. 2010, Hanson et al. 2013). These asexuals engender important evolutionary questions regarding evolution of meiosis-specific genes following asexuality, and whether the retention of at least some components of meiosis can allow asexual lineages to clear harmful mutations and increase the efficacy of selection. By comparing the evolution of meiosis genes between sexual vs asexual, 1 and young vs old asexual P. antipodarum I can determine: 1) whether meiotic genes in asexual lineages are in the act of accumulating mutations, which will illuminate whether meiosis is being lost, modified, or maintained, 2) how observed changes to meiotic machinery compensate for transitions to asexuality, and 3) extrapolate how changes to meiotic genes could explain observed patterns of polymorphism and substitution in asexuals. Evolution of Genes under Relaxed Selection I used comparative analysis of the rates and patterns of molecular evolution in meiosis-specific genes in obligately sexual and obligately asexual lineages of the snail Potamopyrgus antipodarum to determine whether genes involved in meiosis evolve under relaxed selection in asexuals. Genes under relaxed selection are genes that accumulate mutations with little to no fitness cost to an organism. Over time, these genes acquire mutations that are expected to result in pseudogenization and subsequent degradation as mutations continue to accumulate (Lynch & Conery 2000, Zhang 2003, Lahti et al. 2009). Pseudogenes then, are the no-longer-functional remnants of functional genes that have experienced a shift from selection to maintain the gene to relaxed selective constraints. The gene-to-pseudogene transition happens when the gene is rendered nonfunctional by one or more mutations (e.g. a frameshift or nonsense mutation) that, because selection is relaxed, can occur with little or no fitness cost to the organism (Li et al. 1981, Gojobori et al. 1982). The pseudogene will continue to accumulate silent mutations until the original functional gene is no longer identifiable. Multiple studies have assessed the fate of genes under relaxed selective constraints following gene duplications (Mungpakdee et al 2008, Sur et al 2013, Yampolsky & Bouzinier 2014,) and after the loss of ancestral characters such as opsin genes in cave fish (Yokoyama et al 1995) or tooth loss in birds (Meredith et al 2014) . These studies indicate the common fate of a gene under relaxed selection is pseudogenization followed by sequence degradation. Even so, degradation and eventual loss of pseudogenes is not always a foregone conclusion. For example, recent studies have suggested that some pseudogenes maintain expression in some organisms (Birney et al. 2007), have roles in negative (Tam 2008) and positive (Guo & Zhang 2011) gene regulation, and may be resurrected to perform a particular function (Chen et al 2011). 2 Genes used only for meiosis are expected to experience relaxed selective constraints in asexuals that do not use meiosis relative to the same genes in sexual counterparts. Some of these meiosisspecific genes are part of a Meiosis Detection Toolkit (MDT) (Schurko & Logsdon 2008) that can and has been used to assess whether there is a potential for meiosis, which is supported by the presence of conserved genes with open reading frames known only to perform vital meiotic functions. I identified genes from this toolkit in the genomes of sexual and asexual P. antipodarum and then used comparative analyses of the rates and patterns of molecular evolution in these genes to: 1) assess their meiotic capability and 2) characterize the type and strength of selection experienced by these genes. This allowed me to determine whether selection was relaxed in asexuals relative to sexuals, and if genes had experienced pseudogenization in the asexuals. This study is the first to my knowledge to assess the genic consequences of relaxed selection for meiosis-specific genes in a natural system comprised of closely related and coexisting sexual and asexual lineages. This study is also novel in its potential to capture meiosis-specific genes in the act of degeneration in recently derived asexual lineages. Meiosis in Sexual Reproduction Sexual reproduction is the process by which distinct genetic material is combined (conventionally by two mating types each contributing respective genetic material) to create genetically unique offspring. This process is accomplished by the production of specialized gametes, with one gamete type produced by each mating type, which fuse to create the initial zygote. A successful mature gamete requires altered amounts of DNA (typically a reduction to about half of the parental genetic material) and is usually genetically unique relative to either parent. Eukaryotes have evolved a highly conserved specialized process of cellular division, meiosis (see Dacks & Roger 1999, Villeneuve & Hillers 2001, Ramesh et al 2005, Malik et al 2008), that creates reduced gametes via a reductional division with novel combinations of genetic material via recombination within progenitor cells (meiocytes). The reduction of ploidy and novel allele combinations within each gamete followed by their subsequent fusion facilitates restoration of ploidy and production of a new genetically unique offspring. Reductional division in meiosis is typically accomplished by one phase of DNA replication at S 3 phase, creating chromatids in meiocytes, followed by two phases of cellular division known as Meiosis I and Meiosis II. Meiosis I results in reduced meiocytes with recombinant chromosomes via the process of crossover. Crossover first requires double-stranded breaks and resection of chromosomes. Single strands of DNA at break points, created by resection, then invade homologous chromosomes to initiate homologous repair by DNA polymerase culminating in Holliday Junctions. After repair, Holliday Junctions are resolved to create recombinant or nonrecombinant chromosomes. Homologous chromosomes then segregate into daughter cells that undergo a second division during Meiosis II separating sister chromatids (reviewed in Zickler & Kleckner 1999). In animals, these daughter cells undergo final developmental stages of spermatogenesis in males (Hecht 1998), oogenesis in females (Sagata 1996), or both spermatogenesis and oogenesis within a hermaphroditic species (Singaravelu & Singson 2011), to create mature gametes. External or internal fertilization facilitates the fusion of mature gametes to complete the process of sexually producing a new individual. Sexual reproduction has been extremely successful, evident in the ubiquity of sex throughout eukaryotes despite its costs (Williams 1975, Smith 1978, Bell 1982). Because males cannot directly produce offspring, the number of individuals with direct reproductive capability in a dioecious sexual population is halved relative to asexuals (typically entirely female). If all else is equal, this so-called "two-fold" cost of sex will result in substantially lower growth rate of the sexual population relative to asexual competitors (Smith 1978). Energetic costs and exposure to predators and parasites during copulation represents additional risks inherent in sexual reproduction (Ronkainen & Yloneh 1994). From a genetic standpoint, recombination confers additional costs via the potential to break up beneficial combinations of alleles at different loci (Muller 1950). Recombination can also result in crossover and chromosomal segregation errors that lead to aneuploidy, which itself often leads to fetus loss, debilitating fetal phenotypes, or serious congenital defects. The existence of these substantial costs and risks associated with sex suggest there must be significant benefits to sexual reproduction that would explain its ubiquity in nature. The predominance of sexual reproduction is attributed to several potential advantages. First, the ability of sexual reproduction to produce and maintain genetic variation through meiotic 4 recombination can increase the efficiency of selection with respect to removing harmful mutations and producing novel combinations of beneficial alleles (Smith 1978, Williams 1975, Barton & Charlesworth 1998, Burt 2000, Connallon & Knowles 2007, Hill & Robertson 2007, Hadany & Comeron 2008). Multiple studies have also shown that the variation generated by sexual reproduction might help organisms escape parasites and diseases (Jokela et al. 2009, Kerstes et al 2012, Mostowy & Engelstaedter 2012, Hogsdon & Otto 2012). Asexual Reproduction Asexual reproduction (parthenogenesis) is a mode of reproduction whereby females produce genetically identical offspring. Asexuals typically avoid meiotic reduction (Suomalainen et al.1976, Bell 1982, Mogie 1988), with the implication that genes used only for meiosis should experience relaxed selection relative to these same genes in sexual relatives. To recapitulate, asexual organisms should outcompete sexual counterparts due to higher rates of population growth, maintenance of linkage between and reliable inheritance of beneficial allele combinations, avoidance of meiotic errors resulting in aneuploidy, and the ability to reallocate resources that otherwise should be needed for copulation or mating behavior to other processes, and avoidance of risks associated with mating behavior like predator attack and disease exposure. Even so, asexual lineages are typically short lived relative to sexual counterparts (Williams 1975, Smith 1978, White 1978, Bell 1982) and seem to be recently derived from sexual ancestors (Neiman et al 2005, Beck et al 2011). Why asexual lineages do not persist as long as sexual lineages remains unclear (Schwander & Crespi 2009), but is often ascribed to various negative genomic consequences of the absence of sex. These consequences include genome-wide linkage disequilibrium which, while efficient at maintaining alleles that are beneficial when linked, requires beneficial allele combinations to independently occur within a single lineage (Kondrashov 1993, De Visser 2007, Desai et al 2007). Lack of recombination as a consequence of asexuality has also been shown to result in mutation accumulation due to an inability to clear harmful mutations from genomes (Lynch et al. 1993, Neiman et al 2010, Henry et al 2012). These findings stress the importance of meiosis, and in particular recombination, in sexual reproduction. 5 Meiosis-specific genes both tend to be conserved in eukaryotes and play an important functional role in sexual reproduction (Villeneuve & Hillers 2001). As such, genes that are important to meiosis (and are part of the Meiosis Detection Toolkit) should be maintained by selection in sexual lineages. Asexual lineages are derived from sexual ancestors and should thus have meiosis genes. Because these genes should experience relaxed selection following the transition to asexuality, I predict that comparisons of sequence evolution in these genes between sexual and asexual lineages will reveal signatures of relaxed functional constraint (e.g. non-synonymous substitutions, nonsense mutations, and frame shift mutations) on meiosis-specific genes in asexuals relative to sexuals. Using the Meiosis Detection Toolkit to Detect Evidence of Relaxed Selection The Meiosis Detection Toolkit (MDT) is a set of core meiosis proteins whose only chronological and spatial expression is within cells undergoing meiotic division, and whose functional roles have been empirically determined to only be in meiotic events such as crossover and recombination (Schurko & Logsdon 2008). Because these genes are only involved in and are absolutely critical to perform meiosis they are deemed meiosis-specific. The MDT is used to determine if an organism is capable of meiosis by virtue of being able to locate homologues of most, if not all, meiosis-specific genes in the organisms genome with an open reading frame. Conversely, the inability to detect either meiosis-specific homologues with an open reading frame or the homologues themselves indicates an inability to perform meiosis as relaxed selection has removed meiosis-specific genes through deletion and/or pseudogenization followed by sequence degradation. The MDT has been successfully applied in a variety of taxa such as Trichomonas vaginalis (Malik et al. 2008), Daphnia (Schurko et al. 2009), choanoflagellates (Carr et al. 2010), arthropods (Schurko et al.2010), mycorrhizal fungi (Rosendahl 2012), and monogonot rotifers (Hanson et al. 2013) indicating the MDT can be successfully applied to search genomes for these meiosis-specific genes, and can be used to infer if organisms are potentially capable of Meiosis. I have applied the MDT to determine the presence/absence of meiosis-specific genes in sexual and asexual lineages of our model system Potamopyrgus antipodarum, and determined the asexual state of sequence degradation relative to sexuals in meiosis-specific genes: Msh4, Msh5, and Spo11. These three genes are crucial in the initiation 6 and resolution of crossover events and proper homologous segregation, making them exemplar meiosis-specific candidates for genes pseudogenizing under relaxed selection in asexuals. Gene pseudogenization is one of several fates meiotic genes may undergo following a change in reproductive mode (Normark et al 2003, Lunt 2008). A signature of pseudogenization is a nearequal occurrence of non-synonymous polymorphism per non-synonymous site (πa) and synonymous polymorphism per synonymous site (πs) across the entire span of the gene. This pattern would suggest relaxed selection because non-synonymous mutations are not being preferentially removed and are nearly equivalent in frequency to neutral synonymous mutations. Frameshift and nonsense mutations are also expected to accumulate as an altered or truncated amino acid sequence would not have a deleterious effect on fitness. A second possible (though likely rare) fate following relaxed selection would be neofunctionalization, or gain of function, of a previously meiotic gene. Elevated levels of πa relative to πs are consistent with this phenomenon, reflecting positive or diversifying selection for amino acid variants. It is also possible that meiotic genes may be maintained by purifying selection in asexual lineages (Charlesworth et al 1993, Som & Reyer 2007, D’Souza et al. 2010). Under purifying selection most non-synonymous mutations will be eliminated from a population, meaning that πa will be relatively low compared to πs. Selection favoring the maintenance of meiotic genes may be generated by the presence of a meiotic step during the asexual production of gametes, e.g. meiotic divisions that occur but afterwards restore ploidy (Marescalchi et al 1993, Liu et al 2007, Sekin & Tojo 2010), or the existence of a secondary function of meiotic genes that is not meiosis-specific (Lydall et al 1996, Matthews et al 1998, Peters et al 2010). I have used the MDT to assess the type and magnitude of selection on meiosis-specific genes in sexual and asexual P. antipodarum and have obtained evidence concerning the degree of relaxed selection in asexual P.antipodarum. I accomplished these goals by quantifying the frequency of πa relative to πs, quantifying the frequency of frameshift or nonsense mutations, and comparing rates of evolution between sexual and asexual lineages using maximum likelihood analysis. I then inferred the selective pressure on candidate meiosis genes by identifying expectations of selective pressures, outlined in the previous paragraph, that were consistent with observed sequence patterns (see Table 1 for summary of modes of selection and their associated patterns). 7 Study System: Potamopyrgus antipodarum Potamopyrgus antipodarum is a freshwater snail native to New Zealand that is well suited for addressing how relaxed selection influences molecular evolution in meiosis genes because obligately sexual and asexual individuals often coexist and compete (Winterbourn 1970, Lively 1987, Lively 1992), allowing for direct and powerful comparisons between sexuals and asexuals (Dybdahl & Lively 1995). While the reliable production of sexual offspring by sexual female P. antipodarum and asexual offspring by asexual female P. antipodarum indicate that reproductive mode is genetically determined (Wallace 1985, Phillips & Lambert 1989), how new asexual lineages are produced from sexual ancestors and the specific mechanism by which asexual P. antiopodarum produce offspring remain unclear. Evidence gathered from allozyme genotyping of different P. antipodarum populations with high or relatively low male frequencies has suggested that populations of high male frequency are sexual, while populations with very low frequencies of males are populations where females reproduce apomictically (Phillips & Lambert 1989). Despite these uncertainties, it is evident that new asexual P. antipodarum lineages have been separately derived from sexual ancestors on multiple occasions (Neiman and Lively 2004, Neiman et al 2011) allowing me to perform multiple experiments comparing sexual and asexual meiotic genes. Because multiple transitions to asexuality have occurred at different times each asexual lineage represents a unique experiment in studying the evolution of meiosis genes under relaxed selection which may show different patterns in different lineages. I have used this study system to sequence a set of core meiotic genes across several sexual and asexual lineages to investigate evolutionary consequences of relaxed selection. 8 Table 1: Expected Sequence Patterns for Corresponding Modes of Selection. Selection Frameshift and Nonsense Mutations π𝑎 π𝑠 Rates of Evolution (relative to sexual lineages) Relaxed Present ~1 Elevated Purifying Absent <1 Proportional Positive Absent >1 Elevated 9 Summary Evolutionary consequences of asexuality on genome evolution and how asexuals utilize their ancestral reproductive machinery is not well understood, despite the light this can shed on the ubiquity, benefits, and limitations of sexual reproduction through comparisons of sexual and asexual genome evolution. I have aimed to illuminate the genomic consequences of asexuality by performing comparative evolutionary analysis of meiosis-specific genes between sexual and asexual lineages of the snail P. antipodarum. By characterizing the selective pressures and evolutionary patterns of meiosis genes in asexual lineages I address the consequences of asexuality on a host of meiosis genes absolutely essential for sexual reproduction, but whose function is not particularly necessary for asexual reproduction. The general lack of necessity for meiosis in asexuality makes meiosis-specific genes powerful candidates for studying genome evolution associated with asexuality because these genes are likely to be either placed under relaxed selection or undergo some modification, non-exclusively resulting in meiosis-specific gene loss or accumulation of non-synonymous mutations. I compared asexual lineages of varying ages to capture the evolution of meiosis-specific genes ‘in the act’, providing novel data regarding the process of mutation accumulation over time in asexually transmitted genomes. In particular, I use molecular and bioinformatics techniques to infer: 1) the type and magnitude of selective pressure on meiosis-specific genes in asexuals, 2) how observed changes to meiotic machinery accommodate the transition to asexuality, and 3) extrapolate how changes to meiosisspecific genes explain observed patterns of polymorphism and substitution in asexual lineages. Based on these results, I will then draw connections between observed evolutionary patterns in meiosis-specific genes and the evolution of meiosis in asexual P. antipodarum. 10 Chapter 2: Materials and Methods Genome Sequencing and Assembly In collaboration with the rest of the P. antipodarum genome consortium, I used high-throughput Illumina sequencing to sequence genomic libraries produced from three asexual P. antipodarum lineages (Kaniere, Waikaremoana, and Poerua) and two sexual lineages (Ianthe and Alexandrina). A lineage is defined as a line of snails descended from a single female. We used the P. antipodarum mitochondrial phylogeny (Figure 1) to attempt to obtain sequence data from lineages representing the range of genetic diversity in the species. This design also allowed us to use comparisons between sexual vs. asexual, and old vs. new asexual lineages to capture patterns of genome evolution across different times following transitions to asexuality. We extracted DNA by dissecting away the head tissues of three snails from each of the five lineages, pooling the heads together within each lineage, and then using a modified phenolchloroform protocol for DNA extraction. Next, we used the Nextera library preparation protocol to create five libraries (one for each lineage), with approximately 200-600 base pair (bp) insert sizes for each lineage. Each of the five libraries was barcoded and run in one lane of Illumina HiSeq. The five barcoded libraries were also pooled and run together in a sixth lane. All samples were pair-end sequenced on the Illumina Hi-Seq, yielding sequence lengths of 101bp in each direction. We used the CLC bio genomics workbench to filer and assemble sequence reads within each lineage to create five genome assemblies (one for each lineage) ranging from ~15x to ~17x coverage. Gene Assembly and Annotation I first created meiotic gene models by using meiotic protein queries from NCBI to locate genome contigs with a matching DNA sequence within each of the five genome assemblies. Proteins of genes with both meiotic and mitotic functions were also used as queries to locate matching genome contigs as a control, since genes involved in meiosis and mitosis are expected to still be 11 Figure 1: Mitochondrial Phylogeny of Potamopyrgus antipodarum. Maximum likelihood tree of concatenated mitochondrial genomes using a T93G model. Sexual lineages are indicated in white, asexual lineages are black. Lineages sampled for genome sequencing are indicated with the name of the lake from which they were sampled. Genomes not sampled for genome sequencing are only indicated by a square. Tree provided by Joel Sharbrough. 12 under purifying selection in asexuals. I computationally extracted matching contigs and then used Sequencher (version 5.2) to assemble contigs from all five P. antipodarum genomes for each specific gene to create one consensus sequence model for each gene. I then aligned each of these models to sequences in the NCBI database to ensure that: 1) all exons in annotated NCBI sequences aligned to meiosis gene models in the correct order, 2) meiosis gene models aligned to their homologous genes in NCBI, indicating no incorporation into models of ectopic contigs from other genes, and 3) meiotic gene models had the best alignments to sequences in NCBI from other protostomes, indicating no foreign DNA contaminants. Next, I annotated each gene model by computationally locating reads in a P. antipodarum transcriptome that aligned to meiosis gene model queries. The transcriptome I used was based on RNA extracted from ovary tissue of reproductively active female sexual and asexual Potamopyrgus antipodarum and sequenced using Illumina technology. I computationally extracted reads from our transcriptome and transferred these reads to Sequencher (version 5.2). I then aligned matching ovary transcriptome reads back to each gene assembly to create annotated gene models with validated intron-exon boundaries. Finally, I focused on annotated meiosis-specific gene models to design PCR primers for each meiosis-specific gene. PCR Amplification and Gel Electrophoresis of Meiotic Genes I designed primers for the PCR amplification of Msh4, Msh5 and Spo11 (see Tables 2, 3, & 4) with ~50% GC content and a melting temperature of ~56○C. Because these genes were to long (~10 kilobases) to clone in one PCR reaction, I performed PCR (Tables 5 & 6) segmentally across ~2,000 base pair overlapping increments along genes (See Figure 2). PCR amplification was performed on DNA from six asexual and three sexual lineages for Msh4, six asexual and three sexual lineages for Msh5, and 13 asexual and five sexual lineages for Spo11, these lineages were ultimately used in comparative analyses. After PCR, I used gel electrophoresis with LE agarose on 10µl of the 37µl of PCR product to determine whether amplified DNA was the expected size, and assess whether ectopic amplification occurred in negative controls that contained all reagents minus P. antipodarum DNA. If negative controls showed no DNA amplification and PCR yielded amplified DNA of ~2,000bp I used low-melt gel electrophoresis to purify the remaining PCR product. I then excised purified PCR product from the low melt gel 13 Table 2: Msh4 PCR Primers. 14 Table 3: Msh5 PCR Primers 15 Table 4: Spo11 PCR Primers 16 Table 5: Stock and Working Concentration of PCR Reagents. Reagent Stock Concentration Working Concentration MgCl2 25 mM 1.9mM dNTPs 10 mM each 0.25mM each 5x 1x 5 U/µL 0.025 U/µL 2.5 U/µL 0.002 U/µL 5x Flexi buffer Pro Taq Pfu Sdd H2O 1.314mM Forward Primer 50 µM 0.8µM Reverse Primer 50 µM 0.8µM 2 ng/µL 2ng DNA 17 Table 6 : Thermocycler Program used in PCR Reactions. Step Temp. (○C ) Time (minutes) 1 95.0 2:00 2 94.0 1:00 3 53.5 1:30 4 72.0 3:30 (or 1 minute per kb) 5 Go to Step 2, Repeat 39x 6 72.0 10:00 7 4.0 Hold indefinitely 18 Figure 2: Gene Models and PCR Strategy for Meiosis-Specific Genes. Annotated gene models created from tblastn searches of genome and transcriptome assemblies. Introns: gray boxes, exons: black boxes, forward and reverse primers: white boxes. Forward primers are distinguished with an ‘F’ followed by a specific number; reverse primers are distinguished with an ‘R’ followed by a specific number. Step-wise amplification of genes shown by white boxes below models, with a letter designation and size. Amplification of Msh4 (figure2a) was done in 7 PCR reactions. Msh5 (figure 2b) was done in 4 PCR reactions and Spo11 (figure 2c) in 2 PCR reactions. 19 and stored gel slices containing amplified DNA of ~2,000bp at 4oC for future use. Cloning of PCR Products I cloned purified PCR products using the Strataclone cloning protocol that I modified by first heating low melt gel slices at 65○C for 10 minutes, until completely melted. Next, I added 0.25µl of vector, 0.75µl of cloning buffer, and 2.5µl of the melted agarose to each cloning reaction (for details on cloning vector see StrataClone PCR Cloning Kit developed by Agilent Technologies). I placed cloning reactions on the benchtop for ~15 minutes to allow integration of PCR into the vector, creating a recombinant plasmid. I then added ~12µl of competent E. coli to each reaction, which were then immediately placed onto ice for 10 minutes and then heat-shocked at 42○C for 45 seconds to transform plasmids into E. coli. Reactions were then immediately placed back onto ice for two minutes to allow the porous membranes of competent E. coli cells to seal. I then placed 125µl of LB medium into each reaction, which were subsequently incubated at 37○C for two hours to allow for E. coli growth; culture plates containing LB medium and Kanamycin were also incubated. After two hours of incubation I plated bacteria onto culture plates containing LB and Kanamycin, and allowed E. coli to grow overnight. Screening of Clones The plasmids I used for cloning contained: 1) a Kanamycin-resistance gene, and 2) a DNA insertion site within a LacZ reporter gene. The incorporation of PCR product into plasmids disrupts the LacZ gene, producing white bacterial colonies that are Kanamycin-resistant. I screened white colonies of E. coli that had grown on culture plates containing Kanamycin using PCR with vector-specific primers to verify that PCR amplified DNA from low melt gels had incorporated into plasmids (Tables 7 & 8). Using gel electrophoresis I determined if PCR screens had amplified DNA with the length of low melt PCR product plus 200 vector base pairs, indicating successful DNA incorporation into the plasmid. Colonies that I screened were simultaneously grown on new culture plates containing LB and Kanamycin so I could extract 20 Table 7: Stock and Working Concentration of PCR Screening Reagents. Reagent Stock Concentration Working Concentration T3 vector primer 50µM 1.25 µM T7 vector primer 50µM 1.25 µM 10 mM each 0.25 mM each 5x 1x ProMEGA MgCl2 25mM 1.25 mM Neb Taq 5 U/µL 0.0125 U/µL dNTPs ProMEGA 5x Flexi Buffer dH2O 0.3736 mM 21 Table 8: Thermocycler Program used in PCR Screening Reactions. Step Temp. (○C ) Time (minutes) 1 95.0 2:00 2 94.0 1:00 3 53.5 1:30 4 72.0 3:30 (or 1 minute per kb) 5 Go to Step 2, Repeat 39x 6 72.0 10:00 7 4.0 Hold indefinitely 22 confirmed recombinant plasmids from white colonies. For each P. antipodarum lineage, I selected ~three bacterial colonies for each PCR reaction to provide higher confidence in sequencing results via independent replications of sequence in multiple clones. Plasmid Extraction and Preparation I transferred clones that tested positive for recombinant plasmids in PCR screens from culture plates into test tubes containing liquid LB medium and Kanamycin that were then incubated at 37○C overnight. Following incubation, I used the Zyppy Plasmid Miniprep Kit and protocol (see Zyppy Plasmid Miniprep Kits by Zymo Research) to extract plasmids from transformed competent cells. Next, I used spectrophotometry to determine the DNA concentration of each plasmid extraction and then submitted plasmids for Sanger sequencing. Processing and Analysis of Sanger Sequences I used Sequencher (version5.2) to process Sanger reads by trimming vector sequence and manually call ambiguous bases. I then used Sequencher to assemble and create separate consensus sequences from Sanger reads of six asexual and three sexual lineages for Msh4, six asexual and three sexual lineages for Msh5, and 13 asexual and five sexual lineages for Spo11. I annotated Sanger consensus sequences by aligning them to meiosis gene models that had been annotated with transcriptome reads and made from genome contigs. I used these annotated Sanger consensus sequences to identify nonsense or frameshift mutations by visually inspecting gene assemblies. Next, I calculated the frequency of nonsynonymous polymorphisms per nonsynonymous site (πa) relative to the frequency of synonymous polymorphisms per synonymous site (πs) for Msh4, Msh5 and Spo11 in sexual and asexual lineages. I began by concatenating the exon sequences within each lineage to generate a lineage consensus for each gene and aligned these consensus sequences with the Muscle algorithm in MEGA (version six) (Tamura et al. 2013). I then used this alignment and DNAsp (version five) (Librado & Rozas 2009) to calculate πa/ πs with a 150bp sliding window with three bp steps for the genes Msh4 and Msh5. Window sizes that were 23 smaller than this did not capture either πa or πs resulting in ratios of zero or undefined ratios, respectively. Window sizes that were larger than this did not provide enough resolution to assess whether selection was relaxed across the entire gene, or at particular sites. In other words, I chose a window size of 150bp with three bp steps because this range gave the greatest number of comparisons between πa and πs, providing a clearer picture as to selective pressures on meiosisspecific genes. Additionally while I obtained Sanger sequence data from six asexual lineages for both Msh4 and Msh5, one asexual lineage was missing a substantial piece of coding sequence for Msh4 and a different asexual lineage was missing coding sequence for Msh5 because of technical difficulties in PCR amplification across the entire gene. I omitted the two asexual lineages missing Sanger sequence data from this analysis so that the same lineages were being compared in Msh4 and Msh5, avoiding any potential elevated πa/πs between these two genes caused by confounding effects of phylogenetic dependence. For Spo11, I used a window size of 30bp and 3bp steps because I only had 318bp of coding Sanger sequence data, making a window size of 150bp too large to obtain any resolution across available coding sequence. I then used MEGA (Tamura et al. 2013) to quantify and compare the rates and patterns of meiotic gene evolution between sexual and asexual lineages. I used IUPAC ambiguity codes to represent heterozygous sites within consensus sequences of each lineage (Table 9). I then used maximum likelihood analysis and a HYK+G model as implemented in MEGA to generate phylogenetic trees of Msh4, Msh5 and Spo11 from consensus sequences that included coding and noncoding sequence. I chose the HYK+G model because it gave the highest bootstrap confidence values when creating phylogenetic trees. I omitted the asexual lineages missing Sanger sequence data for Msh4 and Msh5 from the creation of their respective phylogenetic trees. I did not remove them both from Maximum Likelihood analysis of Msh4 and Msh5 in order to maximize the number of sexual vs. asexual comparisons. This does make sexual vs. asexual rates of evolution between Msh4 and Msh5 less comparable because the lineages in these analyses are not exactly the same, opening the possibility that differentially elevated asexual branch lengths between Msh4 and Msh5 are caused by confounding effects of phylogenetic dependence. However, if selection is indeed relaxed on all meiosis-specific genes in asexuals then all branch lengths should be longer relative to each sexual lineage, regardless of the phylogenetic distribution of lineages within this species. 24 Table 9: IUPAC Degenerate Codes at Heterozygous Sites for Maximum Likelihood Analysis. Only A and G are used here as examples. However the same basic rules applied for all IUPAC degenerate symbols for any given combination of bases at a site. 1 Base1 (Frequency) A(>25%) Base2 (Frequency) G(>25%) Degenerative Symbol R 2 A(75%) G(25%) a 3 A(>75%) a 4 A(>75%) G(<25%) *Independently replicated in another clone G(<25%) *Only present in 1 clone Site 25 A Chapter 3: Results Accumulated Mutations of Sexual and Asexual P. antipodarum I did not detect any frameshift or nonsense mutations in any of the genes that I studied (Table 10). My sliding window comparisons of nonsynonymous relative to synonymous polymorphism (πa/πs) revealed that most sites had a ratio well below one in all lineages, indicating a low rate of retention of nonsynonymous relative synonymous polymorphism. When I compared the cumulative πa/πs of each site across the coding region of meiosis-specific genes asexuals were higher compared to sexuals, however neither estimate of πa/πs met neutral expectations (Figures 3-5). This result could indicate the presence of less efficient purifying selection in asexuals, which has previously been reported in this system (Neiman et al. 2010) as well as other freshwater asexual snails (Johnson & Howard 2007, Crummett et al. 2013). I did find that πa/πs was considerably higher in Msh4 relative to the other genes (Figure 3). Altogether, the outcomes of these analyses are consistent with purifying selection of meiosis-specific genes in asexual lineages. Evolutionary Rates of Sexual vs. Asexual Meiosis Genes A phylogenetic comparison of the rate of evolution of each of the meiosis-specific genes in sexual vs. asexual P. antipodarum revealed that the genes had similar branch lengths in sexuals and asexuals, (Figures 6-8) suggesting that reproductive mode is not affecting the rate at which these genes evolve. The only potential exception to this pattern came from Msh5. While there were relatively long branch lengths for this gene in some asexual vs. sexual lineages (Figure 7), additional comparisons between coding regions of sexual and asexual lineages indicates that the rates of evolution of Msh5 are not different within exons in sexuals vs. asexuals (Figure 9). This implies that polymorphisms driving the elevated Msh5 branch lengths in some asexuals are predominantly in introns that are selectively neutral, as opposed to asexual polymorphisms in Msh5 exons that show similar evolutionary rates to sexuals. Since there is an a priori prediction that Msh5 exons in sexuals are under purifying selection my result indicates purifying selection is also acting on the exons of Msh5 in asexuals. 26 Table 10: Open Reading Frames in Sexual and Asexual P. antipodarum. This table shows all genes that have been bioinformatically assembled from genome assemblies, representing work from myself, Cindy Toll, Matthew Wheat, and Keagan Kavanaugh. Genes that have more than two sexual and more than 3 asexual lineages have assemblies from genome contigs and Sanger sequence data. Genes in black background are meiosis-specific; genes in white background have roles in meiosis and mitosis. Numbers show the number of lineages that have open reading frames, with no evidence of either frameshift or nonsense mutations. 27 Figure 3: Msh4 Polymorphism. Comparison of nonsynonymous (πa) polymorphism relative to synonymous (πs) polymorphism in three sexual and four asexual lineages. Two asexual lineages are omitted from this analysis, due to poor coverage for the gene Msh4 and Msh5 respectively. I omitted asexuals in order to make this analysis comparable between the two genes Msh4 and Msh5. For this analysis I used a window of 150bp and 3bp steps, the location of these windows relative to the exons of the gene is indicated below the x-axis. The inset graph shows the total ratio of πa to πs across the entire coding region in sexuals vs. asexuals. The difference between total πa/πs of sexual vs. asexual is not significant (NS) (Fisher’s Exact Test, p >0.05). 28 Figure 4: Msh5 Polymorphism. Comparison of πa relative to πs in sexual and asexual lineages. The same four asexual and three sexual lineages from analysis of Msh4 were compared using a window of 150bp and 3bp steps, the location of these windows relative to the exons of the gene is indicated below the x-axis. The difference between total πa/πs of sexual vs. asexual is not significant (NS) (Fisher’s Exact Test, p >0.05). 29 Figure 5: Spo11 Polymorphism. Comparison of πa relative to πs in both sexual and asexual lineages. For this analysis 5 sexual and 13 asexual lineages were used to compare polymorphism across four exons. Reproductive modes were compared using a window of 30bp and 3bp steps, the location of these windows relative to the exons of the gene is indicated below the x-axis. The difference between total πa/πs of sexual vs. asexual is not significant (NS) (Fisher’s Exact Test, p >0.05). 30 Figure 6: Maximum Likelihood Analysis of Msh4. Branch length of sexual (white) and asexual (black) lineages was determined using an HKY+G model with 100 bootstrap replicates. The numbers correspond to lineage/ploidy identities as follows: 1: Lady (2x), 2: Grasmere (2x), 3: Ianthe Field Collected (2x), 4: Brunner (3x), 5: Okoreka (3x), 6: Kaniere (3x) omitted due to stretches of zero Sanger sequence coverage, 7: Grasmere (3x), 8: Poerua (4x), 9: Gunn (3x). 31 Figure 7: Maximum Likelihood Analysis of Msh5. Branch length of sexual (white) and asexual (black) lineages was determined using an HKY+G model with 100 bootstrap replicates. The numbers correspond to lineage/ploidy identities as follows: 1: Lady (2x), 2: Grasmere (2x), 3: Ianthe Field Collected (2x), 4: Brunner (3x), 5: Okoreka (3x), 6: Kaniere (3x), 7: Grasmere (3x), 8: Poerua (4x), 9: Gunn (3x) omitted due to stretches of zero Sanger sequence coverage. 32 Figure 8: Maximum Likelihood Analysis of Spo11. Branch length of sexual (white) and asexual (black) lineages was determined using an HKY+G model with 100 bootstrap replicates. The numbers correspond to lineage/ploidy identities as follows: 1: Lady (2x), 2: Grasmere (2x), 3: Ianthe (2x), 4: Brunner (3x), 5: Okareka (3x), 6: Kaniere (3x), 7: Grasmere (3x), 8: Poerua (4x), 9a/b: Gunn (3x), 10: Taylor (3x), 11: Te Anau (3x), 12: Brunner (4x), 13: Tarawera (3x), 14: Rotoroa (2x), 15: Ianthe Lab Lineage (2x) . 33 Figure 9: Evolutionary Rate of Sexual and Asexual Msh5 Coding Region. Branch length of sexual (white) and asexual (black) lineages was determined using an HKY+G model with 100 bootstrap replicates. The numbers correspond to lineage/ploidy identities as follows: 1: Lady (2x), 2: Grasmere (2x), 3: Ianthe Field Collected (2x), 4: Brunner (3x), 5: Okoreka (3x), 6: Kaniere (3x), 7: Grasmere (3x), 8: Poerua (4x), 9: Gunn (3x) omitted due to stretches of zero Sanger sequence coverage. 34 Chapter 4: Discussion The absence of observed frameshift or nonsense mutations in meiosis genes of asexual P. antipodarum suggests that these mutations are deleterious and selectively removed from populations before they can be detected. Additionally, the low frequency of πa relative to πs in sexuals and asexuals indicates that selection is removing nonsynonmous polymorphisms. Finally, maximum likelihood-based comparisons of evolutionary rates between sexual and asexual lineages revealed that these meiosis-specific genes evolve at similar rates in sexual and asexual P. antipodarum. Because meiosis genes in sexual organisms are expected to experience purifying selection, the similar rates of evolution in sexual and asexual P. antipodarum suggests that purifying selection is also acting on asexuals. My results can be interpreted in several ways: 1) Asexual P. antipodarum are engaging in cryptic sex that has yet to be observed, 2) Asexuals P. antipodarum are automictic, and 3) Asexual lineages of P. antipodarum are so recently derived that relaxed selection has not yet translated into a discernable increase in the rate of mutation accumulation in meiosis-specific genes relative to sexual counterparts. The remainder of my discussion will aim to address these possibilities in light of what is known about P. antipodarum, meiosis in automictic asexuals, and evolution of genes under relaxed selection. Several studies have used molecular tools to detect evidence of cryptic sex in organisms previously thought to be obligately asexual (Villate et al. 2010). Similar studies of microsatellite (Weetman et al. 2002) and minisatellite (Hauser et al. 1992) inheritance in P. antipodarum, however, have found no evidence for recombination or gene flow in asexual P. antipodarum. Additional lack of evidence for recombination in this system comes from allozyme genotyping of natural P. antipodarum populations that are completely or almost completely female with low male frequencies <10%, to populations that have higher male frequencies of 20-50% (Winterbourn 1970, Lively 1987, Wallace 1992, Lively & Jokela 2002). These low male populations have been found to almost exclusively reproduce by apomictic parthenogenesis, while the higher male populations usually reproduce sexually (Phillips and Lambert 1989). The absence of evidence for recombination and gene flow in asexual P. antipodarum suggests that it is unlikely that selection is maintaining meiosis-specific genes that enable asexual P. antipodarum to at least occasionally engage in canonical sex. It is also possible that the apparent maintenance of meiosis-specific genes in asexual P. antipodarum reflects automixis, whereby 35 gametogenesis in asexual organisms incorporates components of the meiotic process preceded by premeiotic endoduplication coupled with endomitosis or followed by gamete fusion, to restore ploidy (Marescalchi et al. 2003, Lampert et al. 2007, Mogie 2013). The possibility of automixis is again confounded by the lack of evidence for recombination in asexual P. antipodarum. Asexual P. antipodarum do on rare occasions produce males (Neiman et al. 2012); these males produce morphologically normal but often aneuploid sperm via an apparently modified meiotic process (Soper 2013). The fact that these males do seem to use some elements of meiosis suggests that there may be retention of meiotic processes in their asexual female mothers. The production of rare asexual males also raises the possibility of contagious asexuality (Sandrock et al. 2011, Jaquiery et al. 2014). Infectious asexuality has been heavily studied in parasitoid wasps (Sandrock & Vorburer 2011), aphids (Jaquiery 2014), daphnia (Paland et al. 2005, Sandrock et al. 2011), and rotifers (Tucker 2013). Contagious asexuality involves a recessive meiosis-suppressing asexual allele that segregates in the sexual population, until a homozygous female is produced with the asexual phenotype, starting the first asexual lineage. An asexual lineage will continue to reproduce and on rare occasions bear males. These asexually-produced males will be able to fertilize sexual females and as such, transmit the asexuality-inducing allele to the offspring, initiating a new asexual lineage. Contagious asexuality is supported in P. antipodarum because the variable ploidy across asexual P. antipodarum sperm suggests some components of meiosis are suppressed, possibly via a meiosis-suppressing element that induces the asexual phenotype. Additionally, asexually-produced male P. antipodarum make sperm that appear morphologically similar to sexual males and will copulate with sexual females (Soper et al. 2015 in press), which supports rare asexually-produced males transmitting their meiosis-suppressing allele to a sexual females egg. The elevated ploidy in asexual P. antipodarum supports the possibility of aneuploid sperm fertilizing a sexual euploid egg, and the multiple independent transitions to asexuality in P. antipodarum are consistent with rare asexually-produced males repeatedly fertilizing sexual females to found new asexual lineages. The final possibility for why meiosis-specific genes in asexual P. antipodarum do not appear to be degrading is that selection is relaxed in asexual P. antipodarum that are too young relative to other ancient asexual taxa who have experienced asexuality long enough to have accumulated 36 more nonsynonymous mutations in their meiosis-specific genes than their sexual counterparts (Pellino et al.2013). Other studies that have evaluated meiosis gene evolution in asexual plants (Pellino et al. 2013), degradation of eyes in cave fish (Yokoyama et al. 1995), and degradation of teeth in birds (Meredith et al. 2014) have found elevated rates of nonsynonymous relative to synonymous substitutions, as well as frameshift and nonsense mutations, in genes that are exclusively associated with traits whose ancestral function are no longer relevant to that organisms fitness. Selection on these ancestral traits has been relaxed in these systems for millions of years. By contrast, the oldest asexual P. antipodarum lineage is only around 500,000 years old, suggesting that asexual P. antipodarum may simply be too recently derived from sexual ancestors for the molecular signatures of relaxed selection to become apparent. 37 Chapter 5: Summary Comparing evolutionary patterns between sexual and asexual P. antipodarum provides a powerful means of evaluating the evolutionary consequences of asexuality. By sequencing and performing comparative analysis of meiosis-specific genes between sexual and asexual lineages of P. antipodarum, I have provided evidence that meiosis-specific genes in asexual P. antipodarum are not in the process of degrading. Potential explanations for this surprising result include the relatively recent derivation of most asexual P. antipodarum lineages or because asexuals are in some way utilizing meiosis genes. 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