Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
-EVOLUTION OF ASEXUALITY IN INSECTSPOLYPLOIDY, HYBRIDIZATION AND GEOGRAPHICAL PARTHENOGENESIS -MAGNUS LUNDMARK- DEPTARTMENT OF MOLECULAR BIOLOGY UMEÅ UNIVERSITY Evolution of asexuality in insects -polyploidy, hybridization and geographical parthenogenesis Magnus Lundmark Umeå, 2007 Department of Molecular Biology Umeå University SE-901 87 Umeå Sweden Akademisk avhandling Som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet för erhållande av filosofie doktorsexamen i genetik framlägges till offentlig försvar i Major Groove (byggnad 6L), fredagen den 16e februari 2007, klockan 13.00. Avhandlingen kommer försvaras på engelska. Examinator: Fakultetsopponent: Professor Åsa Rasmuson-Lestander Docent Davis. E. Parker, University of Aarhus, Inst.of Biol. Sci.; Genetik og Økologi, Aarhus, Denmark. Organisation Document Name Umeå University Doctoral dissertation Dept. of Molecular Biology SE-901 87 Umeå Date of issue Sweden January 2007 Author Magnus Lundmark Title Evolution of asexuality in insects -polyploidy, hybridization and geographical parthenogenesis Abstract Asexual reproduction and polyploidy are relatively rare in animals with chromosomal sex determination and always represent a derived condition. To accomplish asexual reproduction several changes in gene expression are required in the mechanism of oogenesis. Polyploidy increases the cell volume and also gives rise to alterations in general physiology. Nevertheless, there are asexual animals that not only survive but seem to be doing better than their sexual progenitors. This is expressed in the distribution pattern called geographical parthenogenesis. Using molecular phylogeny, I here examine the evolution of Otiorynchid weevils, mainly Otiorhynchus scaber and sulcatus in an attempt to trace the evolutionary history and find out what causes the variation in success of different parthenogens. I also evaluate the contribution of asexuality, hybridity and polyploidy as explanations behind geographical parthenogenesis in insects. I conclude that what is called O. scaber is, in fact, a set of geographical polyploids as polyploidy and not asexuality explains the difference in clonal success. I also argue that O. sulcatus is a recently formed clonal species of non-hybrid origin that may well be a good example of a true general purpose genotype. I find little support for asexuality or a hybrid origin as explanations behind geographical parthenogenesis in insects. Finally, I argue that polyploidy in all eukaryotes should be seen as an opportunity for the species evolution, not as a limitation that ensures the demise of the taxa. Key words: Otiorhynchus, weevils, geographical parthenogenesis, asexual, hybridization, polyploidy Language: English ISBN: 978-91-7264-257-7 Number of pages: 63 + 5 papers Signature: Date: 2007-01-15 Sammanfattning Även om sexuell förökning är det som absolut vanligast bland djur och växter så finns det många arter som har återgått till att föröka sig klonalt. Bland dessa asexuella arter så är ett utbredningsmönster kallat geografisk partenogenes vanligt (GP). Arter med GP har både sexuella och asexuella former. De sexuella hittes ofta i centrum av artens utbredning medan de klonala är spridda runt omkring eller på högre latitud eller altitud. De klonala formerna har oftast en större utbredning och ekologiskt framgångsrika. Orsaken till denna förvånande framgång hos dom klonala formerna debatteras dock. De klonala formerna är ofta inte bara asexuella, de har ofta uppkommit genom hybridisering mellan olika arter och är ofta också polyploida (har fler än två kromosomuppsättningar). Både hybridursprung och polyploidi är möjliga kandidater som förklaring till de klonala formernas succe. Jag har inom denna avhandling undersökt vad som orsakar distributionsmönstret GP hos insekter. Jag har fokuserat särskilt på öronvivlar (skalbaggar av släktet Otiorhynchus) och arten O. scaber. Jag har undersökt deras evolutionära historia med hjälp av gen-baserad släktskapsanalys. Jag har även undersökt vilka slutsatser man kan dra av att ta hänsyn till alla kända insektsarter som rapporterats ha GP. Mina slutsatser är att asexuella öronvivlar (O. scaber) har uppkommit oberoende flera gånger och att det är deras polyploidi, inte klonalitet, som gör dom framgångsrika. Jag finner även att det saknas bevis för att klonal förökning skulle förklara utbredningsmönstren hos andra insekter med GP. Istället är det troligt att hybridursprunget och famför allt den högre kromosomuppsättningen ger dom asexuella formerna sin framgång. Copyright © Magnus Lundmark Printed by: Solfjädern Offset AB, Umeå 2007 Table of contents Included papers 1 Preface 2 Glossary 3 Introduction 5 Evolution 5 Sexual reproduction 5 Asexual reproduction 9 Automixis 12 Apomixis 13 Pseudogamy 13 Polyploidy 14 Autopolyploidy 15 Allopolyploidy 16 Geographical parthenogenesis 16 Evolutionary models 19 Tangled bank hypothesis 20 Red Queen hypothesis 21 Metapopulation hypothesis 21 Frozen niche variation model 22 General purpose genotype hypothesis 22 Hybridization hypothesis 23 Polyploidy hypothesis 23 Otiorhychus 26 Otiorhynchus scaber 26 Otiorhynchus sulcatus 29 Aims of the thesis 32 Methodological overview 33 Results 35 Discussion 41 Otiorhynchus scaber 41 Otiorhynchus sulcatus 42 Sexual and asexual reproduction 43 Geographical parthenogenesis and Sexuality vs. Asexuality 44 Asexuality vs. Hybridity vs. Polyploidy 46 Conclusions 50 Acknowledgements 51 Musings of the author 52 References 53 Papers included in this thesis I. Stenberg P, Lundmark M, Knutelski S, Saura A. 2003. Evolution of clonality and polyploidy in a weevil system. Molecular biology and evolution 20: 1626-1632. II. Stenberg P, Lundmark M, Saura A. 2003. mlgsim: a program for detecting clones using a simulation approach. Molecular ecology notes 3: 329-331. III. Lundmark M. 2006. Polyploidization, hybridization and geographical parthenogenesis. Trends in ecology and evolution 21: 9. IV. Lundmark M, Saura A. 2006. Asexuality alone does not explain the success of clonal forms in insects with geographical parthenogenesis. Hereditas 143: 24-33. V. Lundmark M. 2007. Otiorhynchus sulcatus, an autopolyploid general-purpose genotype species? Submitted manuscript Papers are printed with permission of the publishers: I. Oxford University Press II & IV. Blackwell Publishing Inc. III. Elsevier B.V. 1 Preface Ever since a student destined to be a Charles Robert minister of the gospel for the Church of Darwin England, read a book about stones by (1809-1882) Lyell and went on a sea journey, the Detail of watercolor by science of evolution has evolved. In George 1859 Darwin published "On the origin of Richmond, 1840 species by means of natural selection, or the preservation of favoured races in the struggle for life" and the contemporary Victorian society considered it preposterous (that Man, Ape and Dog all have a common ancestor was hard to accept). Today evolution is a theory so sound and well supported that it is bordering fact, despite some areas still left to explore, and yet the ‘Victorian society’ continues to consider it preposterous. PS. Much homage should be paid to Alfred Russel Wallace for his contemporary findings leading to the same conclusions as Darwin, and for maybe being even more skillful in ‘pissing off’ creationists. 2 Glossary (adapted from Per Stenberg’s thesis ‘Origin and evolution in parthenogenetic weevils’) • Apomixis or apomictic parthenogenesis. In this mode of parthenogenesis an egg develops through mitosis instead of meiosis. Offspring are exact copies of their mother. • Automixis or automictic parthenogenesis; parthenogenesis where the eggs undergo meiosis but the chromosome number of the mother is restored by fusion of the products of meiosis, or through premeiotic doubling of the chromosome number. • A clone; offspring produced through mitosis, in the strict sense through apomictic parthenogenesis. • Homologous; organs, structures or DNA sequences that are similar because of a common evolutionary origin. • Meiosis; mode of cell division associated with the production of gametes in eukaryotes. A diploid cell, with two copies of each chromosome divides twice. The end result is four haploid cells with only one copy of each chromosome. The four cells are in males four sperms and in females one egg and three polar bodies (that usually degenerate). • Mitosis; division of a cell that produces two daughter cells, each with a chromosome number identical to the parent cell. The daughter cells are, except for random mutations, copies of each other. • Monophyletic; is a group which contains all the descendants of a common ancestor, the group has a common ancestor unique to itself. • Mutation; a heritable change in the genetic material that is not caused by the segregation or recombination process. • Nondisjunction; disturbed segregation of chromosomes during either mitosis or meiosis. 3 • Paraphyletic; a paraphyletic group contains some, but not all, of the descendants from a common ancestor. • Parthenogenesis; development of a new individual without fusion of gametes. • Polyploidy; increase in genome number to more than diploid trough the addition of one of several haploid chromosome sets. Triploid is three times and tetraploid is four times the haploid chromosome number. o Autopolyploid; the different chromosome sets belong to a single species o Allopolyploid; they belong to different species. Allopolyploids are often the result of hybridisation. o Endopolyploid; only some tissues of the body are polyploid, while the rest is diploid. • Polyphyletic; groups are formed when two lineages convergently evolve similar character states. Organisms classified into the same polyphyletic group share phenetic homoplasies as opposed to homologies • Pseudogamy; parthenogenetic reproduction, but here the egg division needs to be triggered off by sperm but the nuclei do not fuse and the male genome is not transmitted to the offspring. • Tharavul; a servant of the vulcan species (Vulcanis major) who has voluntarily removed his telepathic powers through surgery in order to better serve his master. Also a somewhat affectionate nickname of one of Prof. Saura’s former students. 4 Introduction Evolution Evolution is the corner stone of modern biology. It consists of a systematic change in the gene pool of a population over time rather than phenotypic change. Populations evolve in that mutations arise in individuals; natural selection favours some of these mutant individuals who become more successful than those that do not have these mutations, and thus the composition of the gene pool of the population change over time. Selection then need not be seen as a deterministic force; what matters is rather the combination of genetic traits that will give the owners of a favourable combination higher fitness, in a certain environment, than individuals with other combinations. Finally, fitness in the sense of evolution is the reproductive yield of a class of genetic variants in a gene pool or population. Sexual reproduction One of the greatest marvels of evolution must be the evolution of sex; Meiosis creating gametes with half the chromosome number of the parents, that fuse after fertilization to restore the chromosome number in the resulting offspring. Sexual reproduction is by far the most common mode of reproduction among animals and plants. Sexual reproduction creates genetical and thus phenotypic variation in offspring (Weismann, 1889; Fisher, 1930; Muller, 1932) which should minimize competition among siblings and allow sexual populations to adapt in a variable environment. 5 Sex is beneficial to organisms in a number of ways, and some of the most important may be: • Merging of beneficial mutations by recombination. Two different mutations in different individuals and different loci on the same chromosome can be brought together in the offspring through recombination (see fig. 1). • Allowing beneficial mutations to escape their surroundings. If a beneficial mutation arises in a deleterious surrounding it may escape through recombination and then not be selected against (Fisher, 1930). • Purging of deleterious mutations. Slightly deleterious mutations will randomly spawn in different genotypes of a population and be hard to purge given that they do not reduce fitness severely. Sex will recombine the genotypes creating offspring with both fewer and more of the slightly deleterious alleles. Synergistic effects among the deleterious mutations will then reduce fitness of the individuals that acquire more of them and the population will be purged (Kondrashov, 1988; Kondrashov, 1993). • Resistance to predators and parasites, i.e. ‘the Red Queen’. Sexual organisms are thought to constantly evolve in order to stay ahead in an evolutionary arms raise between e.g. host and parasite (van Valen, 1973; Hamilton, 1980; Hamilton et al., 1990). Any variation that gives a selective advantage in one species will lead to increases in fitness. However, as different species commonly are coevolving, fitness increase in one species implies that it will get an advantage over the other species, and then acquire more of the resources available to both. Increase in one leads to a decrease in the other and 6 the only way to stay in place in relation to each other is to keep evolving. "It takes all the running you can do, to keep in the same place." - Through the Looking Glass by Lewis Carroll. For reviews about the evolution of sex see (Bremermann, 1980; Kondrashov, 1988; Kondrashov, 1993; Barton & Charlesworth, 1998; Burger, 1999; Otto & Lenormand, 2002) Figure 1: Adaptive mutations that occur independently in locus a and b can be brought together by recombination in a sexual species (A) while in a clonal one (B) they must arise in the same independently in the same lineage to achieve the same genotype. Despite all the above, it is theoretically hard to understand why sex prevails, in fact, it has been termed “the queen of problems in evolutionary biology” (Bell, 1982). If you compare asexual and sexual reproduction, you find that clonal females should have twice the reproductive output than sexual ones, given that everything else is equal (Williams, 1975; Maynard Smith, 1978). Maynard Smith (1978) called this the ‘two-fold cost of sex’ (fig. 2). Put simply, it is the cost of producing males. In addition, sexual 7 organisms have to spend energy to find a mate and sexual selection commonly favour traits that reduce the survival of individuals (Maynard Smith, 1978). Figure 2: The twofold cost of sex (Maynard Smith, 1978). Given that other conditions are equal, an asexual female that produces only female offspring will have twice the fitness of a sexual one that produces both male and female offspring. Because of this, an allele that confers asexuality will double its representation in the population each generation. As an asexual female can produce twice as many daughters as a sexual female the ratio of asexual to sexual females should double at each generation. In sympatric populations where the only thing that differs between the sexual and clonal forms is mode of reproduction, it should only be a matter of time before the sexual ones go extinct (fig. 3). Despite this apparent paradox, it is obvious that sexual reproduction has an advantage over asexuality simply because it is the prevailing mode of reproduction among eukaryotes 8 Figure 3: Starting with a single specimen and given a certain carrying capacity of the environment, a clonal form of a species will replace a sexual population in a number of generations depending on the starting size of the sexual population, all else being equal. (Lively & Lloyd, 1990) Asexual reproduction Asexual reproduction is found all across the 'tree of life' and can be found in most plant and animal groups (excluding mammals). It has originated independently many times (as reviewed by Normark, 2003) and takes a variety of forms. The methods entailed in clonal propagation may vary; hydras reproduce by budding, sponges form gemmules, planarians use fragmentation, echinoderms regeneration and plants may have bulbs, runners, rhizomes or parthenogenesis, and all of them result in offspring that are more or less genetically identical to the parent. However, when you talk about clonal reproduction concerning animals most people refer to some kind of parthenogenesis or thelytoky where females produce only daughters and pass on only maternal genes. In the rest of this thesis I will use 9 clonality, asexuality and parthenogenesis as synonyms for animal parthenogenesis unless otherwise specified. Asexuality has been found in over eighty vertebrate taxa from 14 families of fish, amphibians and reptiles (Alves et al., 2001), it is however most common among insects where over 900 species are reported to be asexual or have asexual forms (Normark, 2003). Asexual animals are commonly viewed as evolutionarily inferior, and short-lived in an evolutionary sense (White, 1970; Bell, 1982), and have mainly been studied as a curiosity or as a contrast in order to solve the puzzle of the evolution of sex (e.g. Muller, 1932; e.g. Hamilton, 1980; Kondrashov, 1988; Hamilton et al., 1990; Turgeon & Hebert, 1994; Ridley, 1995; Jokela et al., 1997; West-Eberhard, 2005). Reproduction without meiotic recombination results in offspring that will be identical clones except for random mutations. This is thought to make asexual lineages vulnerable to parasites and competitors in general in comparison to sexuals (The Red Queen hypothesis; van Valen, 1973) and, in the long run, accumulation of deleterious mutations should decrease fitness to the point where the animal is no longer viable (Muller's ratchet, Muller, 1964). Another detrimental aspect of asexuality may be that as all members of a clonal species more or less share the same genome they should have the same reaction norm which results in high sibling competition and low adaptability (see however Adams et al., 2003; Kumpulainen et al., 2004). Asexual reproduction may be advantageous in that it allows beneficial combinations of characteristics to continue unchanged. A clonal lineage that is well adapted to its surroundings stay adapted while beneficial gene combinations may be broken up in recombining sexual species. Colonization ability in clonal species is also higher than in sexual ones as a 10 single female may initiate a new population. Clones do not suffer from inbreeding and lack the trouble of finding mates, which may be some of the largest problems for sexual animals following a colonization event. When a new population is founded, the twofold reproductive output will also rapidly increase the population size, making subsequent colonization by competitors harder (Adamowicz et al., 2002; Stenberg & Lundmark, 2004). Even though so many clonal insect species are known, little is known about the origin of parthenogenetic reproduction (Normark, 2003) and only a few reports explain causes behind the phenomenon (e.g. Dufresne & Hebert, 1997; Normark & Lanteri, 1998; e.g. Delmotte et al., 2003). The molecular mechanisms that cause the transition to asexuality may be summarized into four different modes (Simon et al., 2003): • Spontaneous parthenogenesis (fig. 4), where mutations occur in the genes connected to sex, meiosis or hormone levels (Butlin et al., 1998; Simon et al., 2002). Some insect species also have the innate ability to spontaneously create asexual offspring when they fail to mate, although the success rate appear to be very low (Seiler, 1961; Suomalainen et al., 1987; Lundmark & Saura, 2007). • Hybridization (fig. 4) is maybe the most frequent path to asexuality in insects. It has been shown in e.g. weevils, stick insects and grasshoppers (Honeycutt & Wilkinson, 1989; Law & Crespi, 2002; Stenberg et al., 2003a; Stenberg & Lundmark, 2004). Typically, clones with a hybrid origin have high heterozygosity and can be identified by the maternal mitochondrial genome from the maternal species and a combination of both parental species in nuclear genes. • Contagious parthenogenesis (fig. 4), results from incomplete reproductive isolation where hybridization between clonal females 11 and the males of the same or closely related species transmit genes for asexuality from the male offspring of these hybridizations into the sexual species gene pool (Jaenike & Selander, 1979; Innes & Hebert, 1988; Rispe & Pierre, 1998; Dedryver et al., 2001). • Infectious origin (fig. 4), is only observed in haplo-diploid species where intracellular microorganisms as Wolbachia cause male killing, cytoplasmic incompatibility or development of haploid eggs to become females (Stouthammer et al., 1993; Werren, 1997; Weeks et al., 2001; Weeks et al., 2002). Automixis There are two major ways of maintaining the parental chromosome number when reproducing parthenogenetically. One way is automixis (meiotic parthenogenesis), where the eggs are still produced through meiosis (Suomalainen et al., 1987), and the diploid chromosome number is commonly restored by fusing two of the haploid meiotic products. Automictic reproduction may erode heterozygosity at all or most loci, which is detrimental in most diploid outbreeding species (Maynard Smith, 1978). Some automictic species as e.g. bagworm moths (Psychidae) find a way around this through a process called central fusion, in which the two central polar bodies of the second maturation division fuse with each other and give rise to the embryo (Seiler, 1961; Lundmark & Saura, 2007) or premeiotic doubling. 12 Apomixis The other way is to abandon meiosis altogether. In apomictic (or ameiotic) parthenogenesis, there is no recombination of alleles and the eggs are being produced by mitosis. The offspring are 'true clones' of the mother, save for random mutations. In apomicts heterozygosity steadily increases as gene mutations and structural rearrangements occur and the heterozygosity is maintained through the generations (White, 1973; Suomalainen et al., 1987). Pseudogamy or Gynogenesis Here offspring are produced by the same mechanism as in apomictic parthenogenesis but copulation or sperm from a related bisexual species are required to initiate egg development. The sperm enters the egg cell but the nuclei do not fuse and males do not contribute any genetic material to the offspring (Haskins et al., 1960; Suomalainen et al., 1987; Schlupp, 2005). This means of course that pseudogamous species never can escape their hosts or sister species, or invade habitats not inhabited by the host without ensuring their own demise (Vrijenhoek, 1984). 13 Figure 4: Modes of origin of parthenogenesis in animals (Simon et al., 2003). Polyploidy Common bananas, giant redwood trees and normal salmon that we eat are examples of polyploid species. Polyploidy, or the presence of more than two basic chromosome sets per nuclei (Darlington, 1932), is found to occur in about half the natural species of flowering plants (Hieter & Griffiths, 1999) and in 40% of the examined species of palearctic terrestrial earthworms 14 (Casellato, 1987) but is relatively uncommon in animals with chromosomal sex determination (Muller, 1952; Mable, 2004). Polyploidy in animals is known to originate by some different mechanisms; failure of cell division during meiosis (gametic nonreduction), failure of cell division after mitotic doubling (which occurs in both animals and plants), production of unreduced eggs and finally hybridization between species (Otto & Whitton, 2000). Newly formed polyploids commonly face severe problems as disrupting effects due to nuclear and cell enlargement, the tendency of polyploid mitosis and meiosis to produce aneuploid cells and gene regulation instability due to epigenetic changes (Comai, 2005). However, polyploid cells and tissues that are larger and more metabolically active, than their diploid counterparts (Hieter & Griffiths, 1999) may be beneficial and polyploid forms of a species have been observed to be more tolerant to abiotic stress and have wider distribution than their diploid relatives (Bierzychudek, 1985; Casellato, 1987; Van Dijk & Bakx-Schotman, 1997; Adamowicz et al., 2002; Stenberg et al., 2003a; Lundmark & Saura, 2006). Polyploidy also gives, at least theoretically, a new lease of life to asexuals faced with a steady accumulation of deleterious mutations (Lokki, 1976b). Autopolyploidy (intraspecific polyploidy) Polyploids are often divided into different groups depending on how closely related chromosome sets they are made up of (following the terminology of Darlington, 1932). Autopolyploids are polyploids with chromosome sets derived from a single species. They can arise from a spontaneous, naturally occurring genome doubling. Stebbins (1950) showed that most cases reported as plant autopolyploids in fact were of hybrid origin; doing so he 15 effectively deflated the ongoing research at the time. Several cases of verified autopolyploids have, however, been found and verified among animals (e.g. Seiler, 1963; e.g. Lundmark & Saura, 2007) and plants (e.g. Seiler, 1963; Soltis & Soltis, 1993; Van Dijk & Bakx-Schotman, 1997; Soltis & Soltis, 2000; e.g. Borgen & Hultgård, 2003; Yamane et al., 2003; Lundmark & Saura, 2007) since then. Allopolyploidy (interspecific polyploidy) In allopolyploids the different chromosome sets are derived from different species. Animal allopolyploidy is much more common than autopolyploidy, it has been observed in most animal taxa that also reproduce asexually (e.g. Hewitt, 1975; White, 1980; Beaton & Hebert, 1988; Normark & Lanteri, 1998; e.g. Alves et al., 2001; Delmotte et al., 2003; Stenberg et al., 2003a; Kearney, 2005) Between the extremes of auto- and allopolyploids, however, there is a complete range of intermediate types, reflecting the range of genetic variation found in the taxon or taxa that gave rise to the polyploid. Geographical parthenogenesis Clonal organisms that are incapable of recombination should have very homogenous populations, but they still commonly show remarkably high levels of genetic diversity (Bell, 1982; Suomalainen et al., 1987; Cywinska & Hebert, 2002). Most known clonal animals also have high ploidy levels (Suomalainen et al., 1987) and/or heterogeneity in their karyotype structure (Judson & Normark, 1996). Furthermore, even if they may have a relatively short evolutionary lifespan, they are often ecologically successful. Despite 16 the recent origin of most asexuals (Avise et al., 1992), there are several cases where asexual organisms have more or less out competed their sexual ancestors (e.g. Lynch, 1984; Suomalainen et al., 1987). The observation that sexuals and clones of closely related species often have different geographic distributions was made by Vandel (1928) and termed ‘geographical parthenogenesis’ (GP). GP interpreted in it broadest sense claims that asexuals more commonly are found in higher altitudes or latitudes, on islands or in island like habitats, in xeric, extreme, stressful, transient, disturbed or marginal habitats than sexual sister species (Cuellar, 1977; Glesener & Tilman, 1978; Bell, 1982; Lynch, 1984; Bierzychudek, 1985; Suomalainen et al., 1987; Cuellar, 1994). One might ask what manner of habitat is left? A more modern and practical interpretation, connected to the question of the evolutionary potential of clones, is that in species with GP the sexuals are found within a small central area, outside of which only clones exist (fig. 5). The clonal distribution is often shifted to high altitudes or harsh climates (Kearney, 2005; Lundmark, 2006; Lundmark & Saura, 2006). This pattern is found in both plants and animals all over the world. Examples include dandelions (Asker & Jerling, 1992), moths (Seiler, 1961) and many weevil species in Europe (Suomalainen et al., 1987) and grasshoppers and automictic lizards in Australia (Hewitt, 1975; Moritz, 1983). 17 Figure 5: An example of geographical parthenogenesis; in the outer areas of distribution clonal forms of the species are found (grey colour). In the central area of distribution only sexual forms exist (black colour). The scenario assumes that clonal forms, or sister species, have originated from the sexual ones (Stenberg et al., 2003a). If different levels of ploidy exist in the clonal forms of the species, the extent of distribution may vary with ploidy level and there can be overlapping distribution between sexuals and different ploidy types. The correlation between ploidy level and extent of distribution observed in, e.g. weevils (Suomalainen et al., 1987; Stenberg et al., 2000; Stenberg et al., 2003a), is not included in Vandel's definition of geographical parthenogenesis (1928; 1940) and we have chosen to call this special form of distribution geographical polyploidy (fig. 6) (Stenberg et al., 2003a). 18 Figure 6. A schematic illustration of geographical polyploidy in O. scaber (Stenberg et al., 2003a); In the central black area sexuals and clones of all ploidy levels exist. In the dark grey middle area triploid and tetraploid clones are found, and in the outer light grey area, corresponding to the widest distribution, only tetraploid clones reside. Evolutionary models Variation in ecological success between sexual and asexual forms in species complexes with GP, and concomitant differences in distribution, is of interest to evolutionary biologists not only to unlock the secret of why sexuality prevails but also because many clonal species have economical value to humans. The explanation to why the variation exists is however a complex issue. As mentioned above, the clones often have a wider distribution and broader ecological tolerance than the sexual forms they originate from (Beaton & Hebert, 1988; Parker & Niklasson, 2000; Schon et al., 2000; Stenberg et al., 2003a). This ecological success of clones has, 19 however, been attributed to aspects of either reproductive mode, elevated ploidy level or hybrid origin by different authors and resulted in a plethora of models. Most of these may, with some naivety, be classified into one of two camps. In the first camp, the mode of reproduction and aspects of a rigid clonal genome is emphasized. These models propose that the distributions are explained mainly by failure of clonal forms to compete with sexuals in the central area, and superior ability of parthenogens to colonize new habitats where they can reproduce rapidly and take advantage of resources. The ‘Tangled bank’, ‘Red Queen’, ‘Metapopulation’ and ‘Frozen niche variation’ -hypotheses are all found in this camp. The models in the second camp instead advocate an adaptive potential of clones connected to a frozen genotype, genomic merger, or elevated ploidy level. They are based on the observation that some clones are more tolerant to abiotic stress (e.g. temperature, desiccation or salinity) and have wider tolerances in what environments and habitats they may utilize in comparison with their sexual ancestors. Unique gene combinations, enlarged body, or cell size and elevated level of heterozygosity are thought to be responsible for these characteristics. Examples are the ‘General purpose genotype’, ‘Polyploidy’ and ‘Hybridization’ –hypotheses. The tangled bank hypothesis The tangled bank model is mainly advocated by Bell (1982). The phrase itself comes from the last paragraph of Darwin’s Origin of Species (1859), in which Darwin referred to a wide assortment of creatures all competing for light and food on a “tangled bank”. According to this concept, any environment where an intense competition for space, food, and other 20 resources exists will favour a diverse set of offspring. As each sibling uses a slightly different niche, all may utilize more resources of the environment than a clone, the members of which are competing for exactly the same resources as its clonal sisters. GP is according to this model a consequence of more complex biotic interactions in the areas where sexuals reside. Clonal organisms, Bell concludes, are not able to compete successfully in those areas and will continously be displaced to surrounding environments (Bell, 1982; Lynch, 1984). The Red Queen hypothesis The Red Queen Hypothesis was put forward by Van Valen (1973). His research suggested that the probability of organisms becoming extinct bears no relationship to how long they already have survived. This is related to GP through an argument that if all organisms continuously have to change and become better in order to survive then clonal organisms are doomed as they create so homogenous offspring. Sexual competitors, predators and parasites will in the central areas adapt and become better and the clones rapidly be out-competed and pushed to surrounding areas (van Valen, 1973; Ridley, 1995). The Metapopulation hypothesis Haag and Ebert (2004) base their hypothesis about GP on population genetics and the fact that asexual organisms does not suffer from inbreeding effects. In the margins of a species distribution populations are commonly small and subdivided. Repeated colonization events with bottleneck effects 21 and low population size will lead to inbreeding depression and low fitness in sexuals. In these areas clones should both be more effective than sexuals at colonizing empty habitat patches and able to invade low fitness sexual populations and drive them do extinction (Haag & Ebert, 2004). The model assumes both that purging of deleterious mutations in sexuals is inefficient so that they actually suffer from inbreeding related low fitness, and that populations are subdivided in the areas where clones are found. The Frozen niche variation model This model is something of a pivotal point between the models that claim that clones are just fugitives and the ones that believe that adaptive benefits may be gained from reproducing asexually. According to Vrijenhoek (1979; 1984) the success of clones in some areas is a consequence of clonal diversity. He proposes that with greater genetic diversity in clonal lineages, the clones are able to capture more of the available micro-niches from sexuals (Vrijenhoek, 1979). Connected to GP this then assumes that the clones are of polyphyletic origin and that multiple asexual lineages coexist creating an ensemble of specialist lineages frozen in different micro-niches or segments that where available to the parent species. Vrijenhoek still views individual clonal lineages as inferior fugitives. The General purpose genotype hypothesis This may be the most inclusive of the models as it may encompass the Frozen niche, Hybridization and Polyploidy –hypothesis (Gade & Parker, 1997). The GPG hypothesis assumes that a generalist clone may survive in 22 areas with specialist sexuals if the resources are not to scarce, and conquer surrounding areas as the members of the clone have a broader niche tolerance than the sexuals (Parker et al., 1977; Lynch, 1984). The production of these generalist clones is facilitated through polyphyletic origins and selection for the lineages that tolerate broad rages of environmental characteristics. I.e. when the environment changes over time, only those clones that may be successful in all of the settings the habitate goes through will persist (Parker et al., 1977). Ergo, over time there will be selection for general purpose genotypes. The Hybridization or heterosis hypothesis Heterosis or hybrid vigour is positive non-additive effects of hybridization. It is observed for example in hybrid corn or broiler chickens, where the hybrid offspring of two inbred sexual lineages become larger and yield better than non-hybrid corn or chicken. In asexuals it has been hypothesised that very high, fixed heterozygosity, may lead to broadly tolerant phenotypes with hybrid vigour (Schultz, 1969; Schultz, 1971; Wetherington et al., 1987). This would, together with novel gene combinations not available to sexuals, be the reason to clonal success in GP complexes according to the hybridization model. The Polyploidy hypothesis Physiological effects as enlarged, body, cell or egg –size, together with having multiple alleles per locus, allow polyploids to become more tolerant to extreme physical stresses and gain unique gene combinations that the 23 sexuals lack explain clonal success and GP according to the polyploidy hypothesis (Vandel, 1940; Suomalainen, 1962; Levin, 1983; Bierzychudek, 1985). Also in this model, heterosis effects are thought to contribute to higher fitness of the clones. Polyploid clones are also thought to be less sensitive to Muller’s ratchet as they possess a more copies of each gene (Lokki, 1976b). The polyploidy and hybridization hypothesis are to large parts overlapping and complementary. Both are separated theoretically from the rest of the explanations of GP as they propose that the importance of reproductive mode is secondary to the effects of hybridization and polyploidy. It is however important to stress that advocates of the three latter models that suggest that GP is due to clonal advantages, not failures, do not disregard the benefits the asexuals receive from being just asexual, e.g. increased colonization ability (Parker et al., 1977; Stenberg et al., 2003a; Kearney, 2005; Lundmark & Saura, 2006). The major complicating factor concerning animal GP is that asexual reproduction, polyploidy and an hybrid origin co-occur in most cases (Lundmark & Saura, 2006). Animal complexes with GP usually have diploid sexuals and parthenogens of a single ploidy level (typically triploids) which links the mode of reproduction to the ploidy level. As the clones commonly also are of hybrid origin all three phenomena may be linked. Because of this, it is troublesome to deduce the factor responsible for the apparent success of clonal forms in these cases. Laboratory corroboration has proven inconclusive and hard to come by (Wetherington et al., 1987; Zhang & Lefcort, 1991; Gade & Parker, 1997), largely due to the nature of clonal samples. Models like the GPG, hybridization and polyploidy -hypothesis, that propose that some clones are more successful 24 than sexuals due to an adaptive benefit of their genome, does not claim that all clonal lineages are superior. Rather, they propose that selection favours those rare clonal lineages that manage to overcome the problems associated with genome duplication and hybrid dysfunction or are lucky enough to receive a ‘multi purpose’ genotype. This mean that it is of little value to state that e.g. synthesised triploids as a group are worse than diploid sexual relatives in regard to a life history trait. Different clonal lineages of the same ploidy level do not comprise a true population as they are genetically isolated from each other, and population means of a trait are thus inapplicable. I.e. selection will not work against one clonal lineage because of another one that is maladapted to the habitat, even if they are sympatric, as they do not share a gene pool. As long as any polyploid lineage shows a higher fitness (i.e. value in the trait measured) in laboratory tests, the study can instead be considered supporting a potential superiority of the clones. It is my belief that although it is not often stated, most if not all authors that work with evolution of clonality, hybridization and polyploidy, be it in plants or animal, assume that the major parts of events leading to these phenomena result in failures due to problems such as gene dosage compensation and cytoplasmic incompatibility. Nature does however supply an ample amount of tries. To separate the effects of reproductive mode, hybridization and polyploidy on ecological and geographical success, it is necessary to study both hybrid and non-hybrid taxa where variation in breeding system and ploidy level are separate. 25 Otiorhychus Almost 20% of all insect species known to reproduce clonally are weevils (Curculionids) (Normark, 2003). All clonal weevils that have been examined so far are apomictics (lack meiosis and recombination) and have chromosome numbers in multiples of 11 (with exception for aneuploids) (Suomalainen et al., 1987). The genus Otiorhynchus has been studied since the beginning of the last century (e.g. Penecke, 1922; Székessy, 1937; Suomalainen et al., 1987). Otiorhynchus contain more than 60 known parthenogenetic forms (e.g. Mikulska, 1960; Suomalainen et al., 1987; Normark, 2003) and most of them are pests on agricultural and forest crops, e.g. the vine weevil, O. sulcatus (Fabricius, 1775), the clay coloured weevil, O. singularis (Linnaeus, 1767) and the strawberry root weevil, O. ovatus (Linnaeus, 1758) (Palm, 1996). Otiorhynchus species with clonal forms very often show geographical parthenogenesis and if several ploidy levels exist, geographical polyploidy. In the species with GP most sexual forms have distributions limited to the eastern Alps (Jahn, 1941) in areas thought to be glacial forest refugia. The refugia are small moist areas that were not glaciated during the last Ice Age, within which different plants and animals are believed to have survived the latest ice ages. Most forest refugia are valleys characterized by a distinct flora that remained free of ice even if the surroundings were glaciated (Ellenberg, 1986) Otiorhynchus scaber Otiorhynchus scaber (Linnaeus, 1758), a small (about 5mm long) flightless weevil that lives most of the year below ground, is the species that has 26 enjoyed the most attention in the population genetic studies. O. scaber is a minor forest pest, mainly on Norway spruce. Larvae feed on the roots of young spruce and adults on both roots and foliage of spruce and blueberry. O. scaber has four different forms, diploid sexuals and diploid, triploid and tetraploid clones (fig. 7) that reproduce by mitotic parthenogenesis. Figure 7: Otiorhynchus scaber of different ploidylevels. From left to right; tetraploid, triploid and diploid females. Tetraploid clones were the weevils that Linnaeus saw and described in 1767. Tetraploid clones are the nominate form of the species, although Linnaeus of course did not know that they polyploids or clones. Sexuals were described first in 1922 by Penecke as a separate species called O. ambigener and Smreczynski (1966) described the triploid form as var. oblongus. The nominal names of the different forms are nowadays not commonly used and I will use the name O. scaber for all the different forms, including the diploid clonal form we discovered in 2003 (Stenberg et al., 2003a). 27 The O. scaber complex shows geographical polyploidy (fig. 8), sexuals and diploid clones are only found in the refugial areas of Austria and Slovenia, sympatric with clones of higher ploidy level (down to the same branch of a single tree) (Stenberg et al., 2000; Stenberg et al., 2003a). Triploid clones are found in the montane and submontane areas of the Alps and tetraploids have conquered most of the Palaearctic spruce forests (Saura et al., 1976; Stenberg et al., 2003a). Figure 8: Geopraphical polyploidy in the O. scaber complex (Stenberg et al., 2003a). Sexuals and all three clonal forms are found in the small central areas (black). Triploid and tetraploid asexuals inhabit the submontane parts of central Europe (dark grey). Tetraploids alone inhabit the full distribution covering most spruce forests in central and northern Europe (light grey). Origin of the asexual O. scaber is thought to result from hybridization events between different subpopulation of the sexuals that have diverged during the separation in different ice age refugia (Stenberg et al., 2003a; Stenberg & Lundmark, 2004). As the genitalia of sexual and clonal females 28 of do not differ (Székessy, 1937), and males copulate with females of all ploidy levels it has been assumed that the clones have originated from the sexuals in this area (Suomalainen & Saura, 1973). Suomalainen (1940) and Seiler (1947) have shown that many parthenogenetic forms of weevils, including O. scaber, have chromosome sets that form separate metaphase plates during meiosis. E.g. in triploids, three separate haploid chromosome sets have been observed, as well as one diploid and one haploid set. This indicates that chance fertilizations by males, of either the same or a closely related species, have caused a stepwise increase in ploidy level (Suomalainen, 1940; Suomalainen, 1969; White, 1973; Lokki, 1976b; Saura et al., 1993). Taken together this is an excellent complex to study evolutionary attributes of asexuality and polyploidy in. Otiorhynchus sulcatus The Black vine weevil, O. sulcatus (Fabricius, 1775) is an all female species with adults that are 9-13mm with dark color and light specs on the elytra (fig. 9). The flightless females reproduce by mitotic parthenogenesis (Seiler, 1947) and all individuals examined are triploid (Suomalainen et al., 1987). The sexual ancestors of the species are not known, nor are any males scientifically documented. 29 Figure 9: Otiorhynchus sulcatus, a species of only triploid clonal females. A common pest on many horticultural and agricultural crops (Moorhouse et al., 1992; Lundmark & Saura, 2007). At the beginning of the 19th century the distribution of the Black Vine Weevil, O. sulcatus, was limited to central Europe. It was observed to be pest by vine growers with a patchy distribution. Nowadays, the weevil is a common pest species with a distribution covering most parts of Europe and North America, parts of Central Asia, South America, New Zealand, Japan etc. (see fig. 10) (Moorhouse et al., 1992). Black vine weevils are now causing serious economical damage as it is very polyphagous and may thrive on a large number of horticultural and agricultural crops. Although the weevil appear to prefer different Grapevine, Strawberry (Fragaria), Rhododendron, Taxus and Cyclamen species, as many as 150 plants have been identified as potential hosts (Smith, 1932; Wanger & Negley, 1976; Moorhouse et al., 1992). The larvae are the main cause of damage as they burrow into and feed on the roots. The adult weevils feed on leaves and fruit and cause mostly ornamental damage, although defoliation is a problem in heavy infestations (Cone, 1968; Moorhouse et al., 1992). The rapid spread and proliferation to new countries and continents is likely due to 30 anthropological activities and huge crop monocultures, but also the fact that Black vine weevils are obligate parthenogens. Recent molecular studies indicate that O. sulcatus has originated trough autopolyploidization and not hybridization as O. scaber (Lundmark, 2007). Figure 10: Approximate distribution of the triploid parthenogenetic Otiorhynchus sulcatus based on data from Moorhouse (1992), internet reports and reports from different horticultural growers (personal communication) (Lundmark, 2007) 31 Aims of this thesis In this thesis my main aim has been to examine if the differences in ecological success and geographical distribution between ‘conspecific’ sexuals and clones, in weevils in particular and in insects in general, really are an effect of the difference in reproductive mode. I have also addressed the following lesser points: 1. What is the evolutionary origin of the different forms of the O. scaber complex? 2. How to reliably identify clonal multilocus genotypes (MLG) when population size, allele frequencies and number of identical observed MLGs are taken into account 3. Polyphagy and host shifts observed in O. sulcatus; is it due to multiple clonal specialists, general purpose genotypes or something else? 4. Is there any correlation between genetical variation and geographical location in O. sulcatus? 5. Are O. sulcatus of hybrid origin or not? During my PhD student period I have additionally worked with the trade-off between asexuality and flight ability in Lepidoptera, but will not defend that in this thesis (Lundmark & Saura, 2007). 32 Methodological overview Working with non-model species is in many regards incredibly rewarding as most of the results you find are interesting and much data actually reveal unknown facts about the study organism. There is however some problem that can be very frustrating and which people working with models may tend to forget. As when your PCR’s does not work and you get the ‘take another marker’ or ‘make new primers’ and you carefully try to explain that all published sequences from the species or even the genera are those the group submitted themselves. Then it is the ever so interesting fact that asexual polyploids are in fact polyploid and maybe also hybrids which means that although mitochondrial data might work as normal nuclear do not. You have to be sure that you compare the right gene copies. If the organism has an inter-species hybrid origin sequence variation will probably be evident on normal agarose gels. If, however, it has been created through hybridizations between different populations of the same species the variation between copies will often be too small to show. The same is true for many polyploids where you want to separate different alleles where variation is to small to result in individual bands, but large enough to result in bad sequence results. Many scientists faced with these problems has turned to sub-cloning where you PCR your marker, clone individual DNA fragments into e.g. Escherichia coli and grow single colonies to multiply it enough to sequence the ‘clean’ copy. In the cases of e.g. hybrid tetraploids it gets intractable rather fast as you do not know how many clones you have to sequence to sample all alleles. You might get a pointer by running the markers on DGGE gels (degradation gradient gel electrophoresis) where very small sequence differences will cause fragments to stop migrating in 33 different parts of the gel more or less uncorrelated to size, but in the end you will still have to do a large amount of sequencing which will limit population studies. The DGGE gels are however a neat tool to verify that you lack intra-individual variation in cases like O. sulcatus where you suspect you are dealing with autopolyploids. If nuclear markers give good sequences directly and no band-variation appear on DGGE’s sub-cloning is blissfully unnecessary. I have in thesis utilized phylogenetic analysis as a tool to construct hypothesises of the evolutionary history of different weevils. The main questions answered by these analyses have been if the beetles are of hybrid origin or not, how many times asexuality has originated, how long ago and of course, how they are related. As large parts of the individuals sampled for the different analysis has been clonal I have tried to avoid over-interpreting the data by testing different methods on the same data, using the conservative cladistics methods as a foundation and then comparing if the results differ with other methods (see e.g. paper I). I would like to point out that unresolved tree topologies do not necessary mean that the data is poor when you study clonal organisms. It is a natural result of including individuals that are highly similar in a number of markers, something that result from bad markers in surveys of pure sexual samples but might be ok or beneficial (as it may reveal clones) in mixed ones. For detailed explanations of the different methods that I have used, I refer to the respective papers. 34 Results Paper I: Evolution of clonality and polyploidy in a weevil system With the use of parsimony analysis and Bayesian inference that where in agreement we obtained a consensus phylogeny that reveals a number of points concerning the Otiorhynchus scaber complex; 1. Tetraploid clonal specimens of a supposed outgroup species, O. nodosus, cluster in one of the two major mitochondrial lineages in the ingroup (fig. 11, lineage B). 2. Austrian and Slovenian sexual specimens cluster in two separate clades with about 5% sequence divergence between them. 3. Asexuality has arisen multiple times in the complex. 4. In the thoroughly sampled sexual populations (Mozirje and Plesch), diploid females cluster in two disjunct groups; e.g. the two groups of diploid females from the Mozirje population sample have accumulated almost 6% sequence divergence since their last common ancestor. Some cluster with the males from their respective sampling site and the rest cluster very distantly in the tree, in clades totally lacking male individuals. 5. Triploids are are obviously polyphyletic. They are found in both major mtDNA lineages and must have originated more than once. 6. All tetraploids are distributed basally in one of the major lineages (B) and are likely also polyphyletic. It is impossible to rule out monophyly, but they have probably originated at least three times. 7. Statistical analysis (see paper III, Stenberg et al., 2003b) based on multilocus genotypes from our allozyme screen of diploid females 35 from Plesch and the two Mozirje samples clearly show the presence of clones. These diploid cryptic clones are limited in distribution to the same refugee areas as the diploid sexuals. Figure 11: The cladistic strict consensus cladogram. Created from the 5,452 most parsimonious cladograms based on three partial mitochondrial gene sequences (COI, COIII, and CytB). Bayesian support values (posterior probabilities) are plotted above relevant internodes, and lowest possible number of evolutionary origins of asexuality as dashed ellipses. Haplotypes submitted to GenBank are marked with an asterisk (*). Identical haplotypes are collapsed into single branches. 36 Paper II: mlgsim: a program for detecting clones using a simulation approach By assessing deviations from Hardy–Weinberg (H-W) expectations it is possible to detect the presence of clonal specimen in a mixed sexual-asexual sample (e.g. Stoddart, 1983). We have used the mathematical formula presented in Parks & Werth (1993). It is used to calculate the likelihood of observing at least n identical multilocus genotypes (MLGs) in a specific sample from a population in H-W equilibrium. ⎡⎛ l N! =∑ × ⎢⎜⎜ ∏ Fai × Fbi i = n n!( N − n)! ⎣⎝ i =1 N Psex ( n ⎞ h⎤ ⎡ ⎛ l ⎟⎟2 ⎥ × ⎢1 − ⎜⎜ ∏ Fai × Fbi ⎠ ⎦ ⎣ ⎝ i =1 ) ( ⎞ h⎤ ⎟⎟2 ⎥ ⎠ ⎦ ) N −n Where n=number of individuals with the same multilocus genotype, N=population sample size, l=number of loci, a=frequency of allele a in the ith locus, b=frequency of allele b in the ith locus and h=number of heterozygous loci. A significantly low Psex-value indicates that the multilocus genotype is a clone and not a result of sexual reproduction. This method has however a problem as it does not say which Psexvalues that is significant and it is impossible to use normal 95% confidence intervals as Psex-values are not true probabilities. In paper II we present our MLGsim software that we developed to calculate significant cut-off values for the Psex test statistica, using a simulation approach. We test it on previously published material and compare our results with the previous ones. 37 Paper III: Polyploidization, hybridization and geographical parthenogenesis In this correspondence paper I discuss the role of polyploidy in general in regard to geographical parthenogenesis and highlight some of the cases where it is incorrect to assign hybrid origins as responsible for the pattern. Paper IV: Asexuality alone does not explain the success of clonal forms in insects with geographical parthenogenesis Paper IV focuses on the insect cases of geographical parthenogenesis where clones reproduce by obligate parthenogenesis, the taxa have chromosomal sex determination and ploidy levels has been verified in both sexual and clonal forms. After excluding internal parasites and taxa with sperm dependent asexuals we survey all published cases of GP in an attempt to evaluate the support for asexuality per se, hybridity or polyploidy explaining the GP distribution. We focus on the few cases with several degrees of ploidy where the distributions of different ploidy forms are well known. Taken together, we find 35 known cases with GP fulfilling our criteria (tab.1, Lundmark & Saura, 2006). Then we count as a single case, or complex, a sexual ancestor and its clonal descendants, no matter if the different forms have nominal species status or not. A total of 20 out of the 35 taxa have a single polyploid clonal form, three have only diploid clones and 12 taxa have asexual forms with more than one ploidy level. In conclusion we find little evidence that asexuality in itself is the main factor behind the success of clones in insect complexes with GP. In all the examined cases about 92% have polyploid clones, 57% only have polyploid 38 clones while a mere 8% only have diploid ones. Only in P. subaptera hybridization and polyploidy may be excluded as explanations for the distribution. Although one should not overlook the relevance of heightened dispersal ability that asexual reproduction gives a colonizing animal, dispersal ability does not seem to be the factor limiting diploid clones in the complexes where distributions of clones with different ploidy levels are known (Seiler, 1961; Adamowicz et al., 2002; Stenberg et al., 2003a). We argue that in the major part of cases the effects of polyploidy or hybrid origin may indirectly select for asexuality. We also observe two clear cases of geographical polyploidy (O. scaber and D. triquetrella) where asexuality directly fails to explain the geographical distribution. Paper V: Otiorhynchus sulcatus, an autopolyploid general-purpose genotype species? In this paper I screen O. sulcatus specimen from different parts of the distribution for genetical variation with one mitochondrial (CO3) and two nuclear markers (one coding, ef1a, and one non-coding, ncnDNA). With the use of specimens from seven other Otiorhynchus species that have both clonal and sexual forms, I make an initial attempt at identifying a close sexual relative to the all clonal black wine weevil. Although this study only includes a small number of specimens, the lack of genetical variation indicates that O. sulcatus is autotriploid, i.e. it has not originated through interspecies hybridization. In none of the specimen examined DGGE revealed haplotype differences between the genomes, consistent with an autopolyploid origin. The low amount of synonymous substitution in ef1a and CO3, and the substitutions in the ncnDNA marker, 39 are consistent with random mutations accumulated in a young clonal lineage. The only patterns in variation connected to distribution that I observe is that the O. sulcatus sampled from United Kingdom are somewhat separated from the rest of the populations. I fail to identify any potential ancestor of O. sulcatus using the phylogenetic analysis of CO3. O. sulcatus is determined to be a sister-group of O. niger (Fabricius) but the amount of synapomorphies grouping the O. sulcatus clade indicate a that the relationship is in a distant past. I find it far more likely that I failed to sample the sexual species that the clones originated from, if they still are extant. I conclude that the lack of genetical variation and extremely polyphagous nature, together with O. sulcatus ability to thrive in a range of different climates indicate that it truly is a general purpose genotype. I can, however, not exclude the possibility that it is the polyploid nature of the weevil that underlies its ecological success, but I can determine that heterosis and a hybrid origin obviously are not necessary for it. 40 Discussion Otiorhynchus scaber With the results from our studies (Stenberg et al., 2003a; Stenberg & Lundmark, 2004) the O. scaber complex has joined a growing number of insect species with clones or GP where asexuality and polyploidy has originated multiple times. We find that asexuality must have arisen at least three times, triploidy at least three and tetraploidy 1-3 times in this species. As sexual lineages may give rise to asexuals and later perish or be missed during the sampling process, ages since clonal lineages has originated may be overestimated and number of evolutionary origins underestimated. With this in mind I find it more likely that both asexuality and polyploidy have evolved in O. scaber more often than what we estimate. We observe several identical MLGs from diploid females sampled in the areas where sexual reside, and conclude that these are clonal. We also find that these diploid cryptic clones must have arisen in connection to hybridization events between different sexual populations of O. scaber that likely has diverged when they have been isolated during the Ice Ages. Given the maternal inheritance of the mitochondrial markers we have used it is not plausible to observe the patterns of clustering between sampling site and genetical belonging that we do, if they are not hybrids (fig. 11). It is however worth to stress that both these patterns and the amount of genetical variation indicate that these hybridization events have to pre-date the latest glaciations. Despite the thorough sampling that this species has been subjected to no diploids have ever been observed outside of the refugial areas where 41 sexuals are found in the Alps, and no triploids have ever been found in the areas where only tetraploids reside (fig. 8). As all examined Otiorhynchus species that are clonal has been noted to reproduce by the same mode of obligate apomictic parthenogenesis, I see no reason to assume that clones of different ploidy levels of O. scaber should differ in the dispersal ability they gain from being clonal. This leads to a direct refutation of asexuality per se in explaining the clear pattern of distribution we observe. Diploids, both sexual and asexual, have failed to spread and proliferate and are limited to the refugial areas where we also find sympatric triploids and tetraploids. Triploids have spread further and share the montane and sub-montane areas of the Alps with tetraploids, and only tetraploids are found over most of Central and Northern Europe (fig 8). It is somewhat alarming to discover cryptic diploid clones among the sexuals in this species that has been studied for decades (e.g. Székessy, 1937; e.g. Jahn, 1941; Mikulska, 1960; Suomalainen et al., 1987; Stenberg et al., 2000) as it raises the question if it is an unusual occurrence or if other weevil species, and maybe even other insect species, harbour similar undiscovered diploid clones. Otiorhynchus sulcatus With the use of immense monocultures we humans shape the environment around us into something that may be very beneficial to clonal pest species. As anthropological activities also facilitate spread of pests over large areas of the world the situation is serious. Taken together with the combination of milder winters and banns on many environmentally hazardous insecticides our knowledge of economically important species needs to increase, as that 42 is what the problems are likely to do. Black vine weevil, O. sulcatus, is an example of a clonal pest species where practically nothing is known about the genetical makeup or evolutionary origin of the species. In just a few decades the species has gone from being a localized minor pest to having an economical impact in large parts of the world (Moorhouse et al., 1992). My initial screen of O. sulcatus from different part of the distribution indicates that this species is of non-hybrid background and has very little genetical variation. Still the weevils go through apparent host shifts and spread into different habitats illustrating extreme polyphagy (Smith, 1932; Cone, 1968; Moorhouse et al., 1992). The success despite lack of genetical variation is in agreement with either the GPG- or the Polyploidy-hypothesis. Given the studies of clonal complexes in the past few years that show non-additive and orchestrated expression of genes in polyploids (e.g. Adams et al., 2003; Auger et al., 2005), I find the black vine weevil a prime candidate to examine in an attempt to evaluate if e.g. epigenetic effects may create phenotypic variation in clones that would explain the polyphagy and host shifts. Sexual and asexual reproduction Sexual reproduction and recombination among genes is prevalent among the majority of plants and animals, of that there is no question, but why it is so is commonly debated. The maybe most established view is that sex functions to provide variation for natural selection to act upon (Weismann, 1889; Fisher, 1930; Muller, 1932) and that recombination rescues the genome from mutational breakdown (Kondrashov, 1988; Kondrashov, 1993). On an evolutionary timescale this appears to be approaching truth 43 even if there are examples of clonal species or even genera that have survived, speciated and even flourished for millions of years, e.g. Darwinulids and Bdelloid rotifers (e.g. Chaplin et al., 1994; Judson & Normark, 1996; Mark Welch & Meselson, 2000). On a shorter timescale, maybe up to a few million years, it is apparent that clonal species may be as successful, or even more so, than the sexual species they are related to. I feel that disregarding the ecological impact of clonal organism because of their relative short life expectancy as a ‘species’ is somewhat dismissive. Even if a clonal lineage will fail to be around in some million years it might undoubtedly leave an imprint on what is actually present at that time as it will change the environment around it and have an impact on competitors, prey, predators and parasites. Especially human food crops and pest on these that often are clonal will via anthropological activities greatly shape the environment around them. With that in mind I find the pattern of geographical parthenogenesis highly interesting as it not only illustrates the adaptive differences between sexual and clonal reproduction, but also secondary attributes of asexuality. Geographical parthenogenesis and Sexuality vs. Asexuality Given that all else is equal a clonal female has a twofold reproductive output compared to a sexual one. This means, as stated before, that asexual organisms should ‘win’ pretty fast in competitive situations, but they do not. Why? Well ‘everything else’ is obviously not equal, and the twofold increase in reproductive output does not appear to actually be twofold (e.g. Seiler & Schäffer, 1960; Roth, 1967; e.g. Enghoff, 1976b; Kumpulainen et al., 2004). Observing the phenomenon of GP the first question I ask myself 44 is; in an ecologically competitive sense, are the clones winning or are they actually loosing? Related to the different hypothesis about GP, do they have adaptive benefits or are they fugitives? The answer I have come to embrace is a little bit of both; The ‘Red Queen’ is certainly realized in species like psychid moths where parasite infestations in the wild have been directly observed to be higher in clonal than sexual specimen (Kumpulainen et al., 2004). It is also likely that the colonization advantage asexuality gives might be important for the survival of clones with fitness lower than that of sympatric sexuals. Further, the Metapopulation hypothesis probably has a lot of merits in explaining the initial dynamics of colonization of pristine habitats as sexuals will suffer from inbreeding and lack of mates, which clones do not. I do not, however, believe that GP at large can be explained by the negative parts of asexuality. Both the Tangled bank and the Red Queen models fail totally to address why sexual species do not expand to the margins of distribution that the clones possess and displace them. I fail to see why e.g. Bell (1982) appears to view the central areas of distribution where sexuals are found as better or more suitable for the species. As long as the clones have a wider distribution and make up a larger part of the species I consider them more successful. Then the ‘fugitive’ models also overlook the observations of overlapping distribution and sympatry of sexual and clonal forms, if the clones always where inferior this should not be able to happen. As reversal to asexual reproduction is problematic concerning e.g. cytoplasmic incompatibility, dosage compensation, erosion of heterozygosity and cytological mechanism involved in the development of eggs, it is likely that the vast majority of clonal lineages that arise will be inferior to their sexual ancestors. Geographical parthenogenesis as a 45 phenomenon however is not to be expected to be a result from the activities of these clones, instead the very few lineages where the sensitive genetical and physiological systems turn out right are more likely to succeed against the opposition and create the distribution patterns. Asexuality vs. Hybridity vs. Polyploidy It is convenient to view the different forms of a species with GP as sexual and asexual, but it is seldom the whole truth. In both animals and plants asexuality is often accompanied by polyploidy and a hybrid origin (Stebbins, 1950; Bierzychudek, 1985; Suomalainen et al., 1987; Kearney, 2005; Lundmark & Saura, 2006). As all three phenomena may convey alterations in phenotype to the organism it is not clear which of them that are responsible for the success of the clones. That asexuals actually have lower fitness, or egg production than sexual relatives (e.g. Seiler & Schäffer, 1960; Roth, 1967; e.g. Enghoff, 1976b) and still have GP indicates that the mode of reproduction cannot be the sole reason to clonal success. Further, there is no intrinsic connection between competitive ability and rate of increase (Roughgarden, 1972; Lynch, 1984). Concerning plants, Stebbins (1950) and Levin (1983) showed that polyploidisation may adapt plants to conditions other than their diploid progenitors are adapted to (de Bodt et al., 2005). Allopolyploids are also expected to show high levels of heterozygosity (Arnold, 1997; Otto & Whitton, 2000; Osborn et al., 2003) which may result in heterosis. In the angiosperms, as much as 47% to 70% of all species are estimated to be polyploid (Masterson, 1994), however, even if all asexual (apomictic) angiosperms are polyploid (Asker & Jerling, 1992) not all polyploid plants are asexual. Concerning GP in plants 46 Bierzychudek (1985) argued that polyploidy was a more parsimonious explanation than asexuality. When all published insect taxa with GP that have chromosomal sex determination and where ploidy level has been verified in both the sexual and the clonal forms are compiled and examined merely three cases out of 35 have only clones with a diploid genome (Lundmark & Saura, 2006). One of these three are of hybrid origin (Warramba virgo), and one is suspected to have a hybrid origin (Nemasoma varicorne). This leaves a single species (P. subaptera) in which hybridity and polyploidy may be safely eliminated as reasons behind the clonal success (Lundmark & Saura, 2006). Although correlation does not always imply causation I consider it striking that in all but one of all the known cases of GP in insects the effects of polyploidy or hybrid origin may be what indirectly selects for asexuality. Although the GPG-hypothesis may encompass the Hybridization and the Polyploidy models, evidence and examples that indicate that the importance of clonality in GP is overrated actually speak against the GPG model as well. If the theory that a general purpose genotype will result in adaptive benefits that will allow a clone to inhabit harsher climates or wider ecological niches are dependent upon heterosis or polyploidy it looses the major part of its justification as a model. The important idea that a GPG clone may in essence ‘remember’ past environmental changes is however still valid even if heterosis or polyploidy would be needed for successful GPG’s to occur (Gade & Parker, 1997). Hybrid origins and heterosis as a stand-alone explanation of GP in insects are not well supported given the 35 cases that we screened. Not really because the data speaks against this hypothesis, but rather as the origins of most cases are unknown. In the few cases where it is known it is 47 also directly connected to polyploidy, with the exception of W. virgo. The D. triquetrella complex also show that geographical polyploidy can occur even if clones are of spontaneous and autopolyploid origin (Seiler, 1961; Seiler, 1963; Seiler, 1964; Seiler, 1965; Lundmark & Saura, 2006). Polyploidy has been considered by many to have a marginal influence in evolution since Muller (1952) and Stebbins (1950; 1971) published their influential works (Otto & Whitton, 2000). Stebbins also claimed that successful polyploids in natural populations almost always are the result of increased heterozygosity accompanying hybridization (Stebbins, 1985). Since then, several cases of autopolyploids have been verified or recorded among animals and plants (e.g. Seiler, 1963; Soltis & Soltis, 1993; Van Dijk & Bakx-Schotman, 1997; Soltis & Soltis, 2000; e.g. Borgen & Hultgård, 2003; Yamane et al., 2003). It has also been noted that autopolyploid hybrid plants show stronger heterosis effects than the corresponding diploid hybrids (Kidwell et al., 1994; Birchler et al., 2003; Auger et al., 2005). Concerning vertebrate evolution, polyploidy is considered to have enabled the evolution of more complex forms of life by allowing new functions to evolve (Ohno, 1970), and logically the same should hold true for other eukaryotes as well, especially insects where there are so many recorded cases (Comai, 2005). The polyploidy hypothesis is by no means likely to explain the success of clones in taxa with GP by itself. Colonization ability, lack of courtship behavior, heterosis and new gene combinations associated with asexuality and hybridity will likely contribute to the circumstances that allow asexuals to prosper. Polyploidy, however, appear to be the common characteristic that in essence all examined cases of insect taxa with GP share (Lundmark & Saura, 2006). If reproductive mode or hybridity were the key phenomena, why do the animals become 48 polyploid? Polyploidisation is a troublesome process in animals with chromosomal sex determination (Muller, 1952) and it is hard to imagine why it would be frequent were it not beneficial. This is evident in animals that lack sex chromosomes where polyploidy need not be tied to asexuality, as plants, or earthworms. Among the terrestrial earthworms that have been examined as much as 40% have been found to be polyploid (Casellato, 1987). Earthworms, such as Lumbricids, may be asexual but are commonly amphigonic (sexual hermaphrodites). The asexuals are in general polyploid, but there are sexual polyploids as well. Geographical parthenogenesis and geographical polyploidy can be studied within a single species that has both diploid and polyploid sexual and asexual forms. For example, Eisenia nordenskioldi, have even ploidy levels (orthoploids) ranging from diploid to octoploid (2n-8n) that are sexual, and a septaploid (7n) parthenogen (Viktorov, 1997). Although the sexual forms cannot self fertilize, and need to find a mate, produce male gametes etc., the distribution of each form is wider the higher the ploidy level (Grafodatsky et al., 1982; Perel & Grafodatsky, 1983; Viktorov, 1997). Polyploidy in asexual species is also important as a buffer against loss of complementation and deleterious mutations. Simulation experiments indicate that clonality can replace sexual reproduction under a wide range of parameters, but only if it is associated with polyploidy (Archetti, 2004) which is in agreement with the conclusions we draw about GP in insects. 49 Conclusions In summary, we find the clonal forms O. scaber complex to be of polyphyletic origin. Both asexuality and polyploidy must have arisen several times. We have discovered the existence of diploid cryptic clones that are restricted to the same areas as the sexuals. From the variation in success of the different clonal forms we draw the conclusion that degree of polyploidy and not asexuality, are main reason behind success in the polyploids and geographical polyploidy. We also note that asexual forms of O. scaber are likely to be of hybrid origin and observe another Otiorhynchid weevil, O, nodosus, that group in the ingroup. Concerning geographical parthenogenesis in insects, we observe little evidence that the mode of reproduction by itself is responsible for the pattern of distribution. We note that the knowledge about the evolutionary origins of parthenogens is too limited to evaluate the role of hybridity and argue that polyploidy are likely to be a more probable explanation. Finally, I observe little genetical variation in different O. sulcatus and argue that they are a young clonal species of non-hybrid origin. The obvious success in the species is most likely dependent of it having a general purpose genotype or polyploid nature. I argue that the pattern of insect species with polyploidy, and incidence in other animals and plants that lack sex chromosomes, is providing surprising proof of the evolutionary potential of polyploids. The ancestral genome duplications that are being revealed through eukaryotic genome projects (see Comai, 2005 for a review) also support that polyploidisation events should be viewed as something that may provide opportunities, not limitations. 50 Acknowledgements My first and major thanks go to my peculiar yet fascinating and inspiring supervisor Anssi. I have found the time as your student to be incredible rewarding in many more ways than just concerning science. I believe that you are part of a special breed of scientists on the verge of extinction and that you are one of the few people with the title who has earned to be called professor. I also would like to thank Anders Nilsson for getting me started on this track and showing me the delights of phylogenetic trees. I owe many thanks to ‘the old Genetics crew’ all of you have shown me what a department can be; especially I thank the old Ph.D. students that introduced me into ‘doktorandhood’. Per, you have been a true friend, an excellent co-worker, a faithful tharavul and somewhat ok as a partner in various crime. I’m still waiting for the invitation to come to Ratan and shoot small cute yummy animals though. Gosia the trustworthy brownie, Dafne the dangerous dreamer aka proud bitch, Lisandro El Toro aka pipett boy, Hans the Green Giant, Mongo, Chronograph-man and many more, and Juan the French the craziest man around (although he’s an honorary member of the Dept.). I would have faced an ELE and perished at the Molbiol without you guys, although it took quite some remodeling to degrade you to the right level of conversation (Juan excluded). Never ever forget the monkey business or the happy bunnies (although I do have exclusive rights). 51 I also thank all the people of the Department that I’ve come to like, Helena, Karin, Mark, Elena, Siw, Åsa, Tord, Mikael and Wicked Wicky and quite a few more. Old Plant Phys, you guys have been great; even if my study organism eats yours you haven’t held too much resentment. I already miss quite many of you and some really good parties. Ola you are super, never ever change, we are BLACK and we are proud (no that has nothing to do with skin melanism, just good taste). Johanna, DM forever (but David needs some chin) and finally Cath the chocoholic, never in my life have I been as surprised about a person as with you; a woman of many levels, starting with Winnie the Puh going through werewolves to a person to be impressed by. My family, Mathisen and my siblings I love you all. Calle and Gustav ‘U are always IMBA’ and I can’t wait to move closer to you. Nanna and Nella and Nalle (lucky it wasn’t Klara, Kerstin and Kalle ;) you mean the world to me even if the frequency of my calls are somewhat low once in a while, and it feels wonderful to know that all my siblings are there for me even if we are not true clones. I would like to finish by giving aeons of thanks to Mojo, minigrisen and trollprinsessan, who makes my life glorious. 52 Musings of the author Evil weevil Why is the weevil supposed to be evil? Almost everyone has heard of the ‘evil weevil’ although most people do not know where the phrase stems from or even what a weevil is. As with many things most people have probably seen them but are not aware of it. Maybe it is because many of those beetles that cause a lot of problems for humans are weevils (why you will get to know if you actually leaf through the thesis). We create huge monocultures that we gather and put in immense stores. To a weevil the acres and acres of cotton just ripe to lay your eggs in must seem like heaven, and moist, tempered indoor greenhouses containing yummy saplings must be a weevil larvae’s idea of a candy shop. Or, it might be because you think they defy the normal order of God and the idea of patriarchal might and right. Females are supposed to mate with males to produce offspring. Anything else is unnatural blasphemy and, even worse, implies that males might not actually be necessary for a species to succeed in our world. No, none of the above I say, the weevils are considered evil because that’s what they are, pure and simple. I mean who wouldn’t be evil if you never got laid. Weevils In Tweed Heads Evil weevil sat at the mall. Evil weevil had a great fall. All the smart piggies, and Allan's men couldn't get weevil back in his pen. - Herbert Nehrlich, 1943 53 References Adamowicz SJ, Gregory TR, Marinone MC, Hebert PDN. 2002. New insights into the distribution of polyploid Daphnia: the Holarctic revisisted and Argentina explored. Molecular Ecology 11: 12091217. Adams KL, Cronn R, Percifield R, Wendel JF. 2003. Genes duplicated by polyploidy show unequal contributions to the transciptome and organ specific reciprocal silencing. Proceedings of the National Academy of Sciences of the USA 100: 4649-4654. Alves MJ, Coelho MM, Collares-Pereira MJ. 2001. Evolution in action through hybridisation and polyploidy in an Iberian freshwater fish: a genetic review. Genetica 111: 375-385. Archetti M. 2004. Recombination and loss of complementation: a more than two-fold cost for parthenogenesis. Journal of Evolutionary Biology 17 1084-1097. Arnold ML. 1997. Natural hybridization and evolution. Oxford Univ. Press, New York. Asker SE, Jerling L. 1992. Apomixis in plants. CRC Press, Boca Raton. Auger DL, Gray AD, Ream TS, Kato A, Coe EH, Birchler JA. 2005. Nonadditive gene expression in diploid and triploid hybrids of maize. Genetics 169: 389-397. Avise JC, Quattro JM, Vrijenhoek RC. 1992. Molecular clones with organismal clones: mitochondrial DNA phylogenies and the evolutionary histories of unisexual vertebrates. Evolutionary Biology 26: 225-246. Barton NH, Charlesworth B. 1998. Why sex and recombination? Science 281: 1986-1990. Beaton MJ, Hebert PDN. 1988. Geographical parthenogenesis and polyploidy in Daphnia pulex American Naturalist 132: 837-845. Bell G. 1982. The masterpiece of nature. Croom Helm, London. Bierzychudek P. 1985. Patterns in plant parthenogenesis Experimentia 41: 1255-1264. Birchler JA, Auger DL, Riddle NC. 2003. In search of the molecular basis of heterosis. Plant Cell 15: 2236-2239. Borgen L, Hultgård U-M. 2003. Parnassia palustris: a genetically diverse species in Scandinavia. Botanical Journal of the Linnean Society 142: 347-372. Bremermann HJ. 1980. Sex and polymorphism as strategies in hostpathogen interactions. Journal of Theoretical Biology 87: 671-702. 54 Burger R. 1999. Evolution of genetic variability and the advantage of sex and recombination in changing environments. Genetics 153: 10551069. Butlin RK, Schön I, Martens K. 1998. Asexual reproduction in nonmarine ostracods. Heredity 81: 473-480. Casellato S. 1987. On polyploidy in Oligochaetes with particular reference to Lumbricids. In: Pagliai AMB and Omodeo P, eds. On earthworms. Modena: Mucchi. 75-87. Chaplin JA, Havel JE, Hebert PDN. 1994. Sex and ostracods. Trends in Ecology and Evolution 9: 435-439. Comai L. 2005. The advantages and disadvantages of being polyploid. Nature Reviews Genetics 6: 836-846. Cone WW. 1968. Black vine weevil larval damage to Concord grape roots at different population densities. Journal of Economic Entomology 61: 1220-1224. Cuellar O. 1977. Animal parthenogenesis. Science 197: 837-843. Cuellar O. 1994. Biogeography of parthenogenetic animals. Biogeographica 70: 1-13. Cywinska A, Hebert PDN. 2002. Origins of clonal diversity in the hypervariable asexual ostracode Cypridopsis vidua. Journal of Evolutionary Biology 15: 134-145. Darlington CD. 1932. Recent advances in cytology. Churchill, London. Darwin C. 1859. On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. William Cloves and Sons, London. de Bodt S, Maere S, van de Peer Y. 2005. Genome duplication and the origin of angiosperms. Trends in Ecology and Evolution 20: 591597. Dedryver CA, Hullé M, Le Gallic JF, Caillaud CM, Simon JC. 2001. Coexistence in space and time of sexual and asexual populations of the cereal aphid Sitobion avenae. Oecologia 128: 379-388. Delmotte F, Sabater B, Leterme N, Latorre A, Sunnucks P, Rispe C, Simon JC. 2003. Phylogenetic evidence for hybrid origins of asexual lineages in an aphid species. Evolution 57: 1291–1303. Dufresne F, Hebert PDN. 1997. Pleistocene glaciations and polyphyletic origins of polyploidy in an arctic cladoceran. Proceedings of the Royal Society of London B 264: 201-206. Ellenberg H. 1986. Vegetation ecology of central Europe. Cambridge University Press, Cambridge 55 Enghoff H. 1976b. Parthenogenesis and bisexuality in the millipede, Nemasoma varicorne C.L. Koch, 1847 (Diplopoda: Blaniulidae). Morphological, ecological and biogeographical aspects. Videnskabelige meddelelser fra Dansk Naturhistorisk Forening 139: 21-59. Fisher RA. 1930. The genetical theory of natural selection. Clarendon Press, Oxford, UK. Gade B, Parker ED, Jr. 1997. The effect of life cycle stage and genotype on desiccation tolerance in the colonizing parthenogenetic cockroach Pycnoscelus surinamensis and its sexual ancestor P. indicus. Journal of Evolutionary Biology 10: 479-493. Glesener RR, Tilman D. 1978. Sexuality and the components of environmental uncertainty: clues from geographic parthenogenesis in terrestrial animals. American Naturalist 112: 659-673. Grafodatsky AS, Perel TS, Radzhabli SL. 1982. Chromosome sets of two forms of Eisenia nordenskioldi (Eisen)(Oligochaeta: Lumbricidae). Doklady Akademii Nauk SSR 282: 1514-1516. Haag CR, Ebert D. 2004. A new hypothesis to explain geographical parhtenogenesis. Annales zoologici Fennici 41: 539-544. Hamilton WD. 1980. Sex versus non-sex versus parasite. Oikos 35: 282290. Hamilton WD, Axelrod R, Tanese R. 1990. Sexual reproduction as an adaptation to resist parasites. Proceedings of the National Academy of Sciences USA 87: 3566-3573. Haskins CP, Haskins EF, Hewitt RE. 1960. Pseudogamy as an evolutionary factor in the poecilid fish Mollienisia formosa. Evolution 14: 473-483. Hewitt GM. 1975. A new hypothesis for the origin of the parthogenetic grasshopper Moraba virgo. Heredity 34: 117-23. Hieter P, Griffiths T. 1999. GENETICS:Polyploidy--More Is More or Less. Science 285: 210-211. Honeycutt RL, Wilkinson P. 1989. Electrophoretic variation in the parthenogenetic grasshopper Warramaba virgo and its sexual relatives. Evolution 43: 1027-1044. Innes DJ, Hebert PDN. 1988. The origin and genetic basis of obligate parthenogenesis in Daphnia pulex. Evolution 42: 1024-1035. Jaenike J, Selander RK. 1979. Evolution and ecology of parthenogenesis in earthworms. American Zoologist 19: 729-737. 56 Jahn I. 1941. Über Parthenogenese bei forstschädlichen OtiorrhynchusArten in den während der Eiszeit vergletscherten Gebieten der Ostalpen. Zeitschrift für angewandte Entomologie 28: 366-372. Jokela J, Lively CM, Dybdahl MF, Fox A. 1997. Evidence for a cost of sex in the freshwater snail Potamopyrgus antipodarum. Ecology 78: 452-460. Judson PO, Normark BB. 1996. Ancient asexual scandals. Trends in Ecology and Evolution 11: 41-46. Kearney M. 2005. Hybridization, glaciation and geographical parthenogenesis. Trends in Ecology and Evolution 20: 495-502. Kidwell KK, Woodfield DR, Bingham ET, Osborn TC. 1994. Relationships among genetic distance, forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations. Theoretical and Applied Genetics 89: 323-328. Kondrashov AS. 1988. Deleterious mutations and the evolution of sexual reproduction. Nature 336 435-440. Kondrashov AS. 1993. Classification of hypotheses on the advantage of amphimixis. Journal of Heredity 84: 372-387. Kumpulainen T, Grapputo A, Mappes J. 2004. Parasites and sexual reproduction in psychid moths. Evolution 58: 1511-20. Law JH, Crespi BJ. 2002. The evolution of geographic parthenogenesis in Timema walking-sticks. Molecular Ecology 11: 1471–1489. Levin DA. 1983. Polyploidy and novelty in flowering plants. American Naturalist 122: 1-25. Lively CM, Lloyd DG. 1990. The cost of biparental sex under individual selection. American Naturalist 135: 489-500. Lokki J. 1976b. Genetic polymorphism and evolution in parthenogenetic animals. VIII. Heterozygosity in relation to polyploidy. Hereditas 83: 65-72. Lundmark M. 2006. Polyploidization, hybridization and geographical parthenogenesis. Trends in Ecology and Evolution 21: 9. Lundmark M. 2007. Otiorhynchus sulcatus, an autopolyploid generalpurpose genotype species? Submitted manuscript. Lundmark M, Saura A. 2006. Asexuality alone does not explain the success of clonal forms in insects with geographical parthenogenesis. Hereditas 143: 24-33. Lundmark M, Saura A. 2007. The trade-off between flight ability and asexuality in moths. Submitted manuscript. 57 Lynch M. 1984. Destabilizing hybridization, general-purpose genotypes and geographical parthenogenesis. Quarterly Review of Biology 59: 257-290. Mable BK. 2004. 'Why polyploidy is rarer in animals than in plants': myths and mechanisms. Biological Journal of the Linnean Society 82: 453466. Mark Welch DM, Meselson M. 2000. Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science 288: 1211-5. Masterson J. 1994. Stomatal Size in Fossil Plants: Evidence for Polyploidy in Majority of Angiosperms. Science 264: 421-424. Maynard Smith J. 1978. The evolution of sex. Cambridge University Press, Cambridge. Mikulska I. 1960. New data on the cytology of the parthenogenetic weevils of the genus Otiorrhynchus Germ. (Curculionidae, Coleoptera) from Poland. Cytologia 25: 322-333. Moorhouse ER, Charnley AK, Gillespie AT. 1992. A review of the biology and control of the vine weevil Otiorhynchus sulcatus (Coleoptera: Curculionidae). Annals of Applied Biology 121: 431454. Moritz C. 1983. Parthenogenesis in the endemic Australian lizard Heteronotia binoei (Gekkonidae). Science 220: 735-737. Muller HJ. 1932. Some genetic aspects of sex. American Naturalist 8: 118138. Muller HJ. 1952. Why polyploidy is rarer on animals than in plants. American Naturalist 59: 346-353. Muller HJ. 1964. The relation of recombination to mutational advance. Mutation Research 1,: 2-9. Normark BB. 2003. The evolution of alternative genetic systems in insects. Annual Review of Entomology 48: 397-423. Normark BB, Lanteri AA. 1998. Incongruence between morphological and mitochondrial-DNA characters suggests hybrid origins of parthenogenetic weevil lineages (Genus Aramigus). Systematic Biology 47: 475-94. Ohno S. 1970. Evolution by gene duplication. Springer-Verlag, Berlin. Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee H-S, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA. 2003. Understanding mechanisms of novel gene expression in polyploids. Trends in Genetics 19: 141-148. 58 Otto SP, Lenormand T. 2002. Resolving the paradox of sex and recombination Nature Reviews Genetics 3: 252-261. Otto SP, Whitton J. 2000. Polyploid incidence and evolution. Annual Review of Genetics 34: 401-437. Palm E. 1996. Nordeuropas Snudebiller. Apollo books, Stenstrup. Parker ED, Jr. , Niklasson M. 2000. Genetic structure and evolution in parthenogenetic animals. In: Singh RS and Krimbas CB, eds. Evolutionary Genetics from Molecules to Morphology: Cambridge Univ. Press. 456-474. Parker ED, Jr., Selander RK, Hudson RO, Lester LJ. 1977. Genetic diversity in colonizing parthenogenetic cockroaches. Evolution 31: 836-842. Parks JC, Werth CR. 1993. A study of spatial features of clones in a population of bracken fern, Pteridium aquilinum (Dennstaedtiaceae). American Journal of Botany 80: 537-544. Penecke KA. 1922. Neue Rüsselkäfer. Wiener entomologische Zeitung 39: 172-183. Perel TS, Grafodatsky AS. 1983. Polymorphism of Eisenia nordenskioldi (Eisen) (Oligochaeta; Lumbricidae). Doklady Akademii Nauk SSSR 269: 1019-1021. Ridley M. 1995. The Red Queen: Sex and the Evolution of Human Nature. Penguin, New York. Rispe C, Pierre JS. 1998. Coexistence between cyclical parthenogens, obligate parthenogens, and intermediates in a fluctuating environment. Journal of Theoretical Biology 195: 97-110. Roth LM. 1967. Sexual isolation in parthenogenetic Pycnoscelus surinamensis and application of the name Pyncoscelus indicus to its bisexual relative (Dictyoptera: Blattaria: Blaberidae: Pycnoscelina). Annals of the Entomological Society of America 60: 774-779. Roughgarden J. 1972. Evolution of niche width. American Naturalist 106: 683-718. Saura A, Lokki J, Lankinen P, Suomalainen E. 1976. Genetic polymorphism and evolution in parthenogenetic animals. III. Tetraploid Otiorrhynchus scaber (Coleoptera: Curculionidae). Hereditas 82: 79-99. Saura A, Lokki J, Suomalainen E. 1993. Origin of polyploidy in parthenogenetic weevils. Journal of Theoretical Biology 163: 449456. Schlupp I. 2005. The evolutionary ecology of gynogenesis. Annual Review of Ecology, Evolution and Systematics 36: 399-417. 59 Schon I, Gandolfi A, Di Masso E, Rossi V, Griffiths HI, Martens K, Butlin RK. 2000. Persistence of asexuality through mixed reproduction in Eucypris virens (Crustacea, Ostracoda). Heredity 84: 161-9. Schultz RJ. 1969. Hybridization, unisexuality, and polyploidy in the teleost Poeciliopsis (Poeciliidae) and other vertebrates. American Naturalist 103: 605-619. Schultz RJ. 1971. Special adaptive problems associated with unisexual fishes. American Zoologist 11: 351-360. Seiler J. 1947. Die Zytologie eines parthenogenetischen Rüsselkäfers, Otiorrhynchus sulcatus. F. Chromosoma 3: 88-109. Seiler J. 1961. Untersuchungen über die Entstehung der Parthenogenese bei Solenobia triquetrella F.R. (Lepidoptera, Psychidae) III. Die geographische Verbreitung der drei Rassen von Solenobia triquetrella (bisexuell, diploid und tetraploid parthenogenetisch) in der Schweiz und in angrenzenden Ländern und die Beziehungen zur Eiszeit. Bemerkungen über die Entstehung der Parthenogenese. Zeitschrift für Vererbungslehre 92: 261-316. Seiler J. 1963. Untersuchungen über die Entstehung der Parthenogenese bei Solenobia triquetrella F.R. (Lepidoptera, Psychidae) IV. Wie besamen begattete diploid und tetraploid parthenogenetische Weibchen von S. triquetrella ihre Eier? Schicksal der Richtungskörper im unbesamten und besamten Ei. Vergleich der Ergebnisse mit F1-Aufzuchten und Beziehungen zur Genese der Parthenogenese. Zeitschrift für Vererbungslehre 94: 29-66. Seiler J. 1964. Untersuchungen über die Entstehung der Parthenogenese bei Solenobia triqutrella F.R. (Lepidoptera, Psychidae) V. Biologische und zytologische Beobachtungen zum Übergang von der diploiden zur tetraploiden Parthenogenese. Chromosoma 15: 503-539. Seiler J. 1965. Untersuchingen über die Entstehung der Parthenogenese bei Solenobia triquetrella F.R. (Lepidoptera, Psychidae) VI. Umbau im Karyotyp der Diploid parthenogenetischen S. triquetrella von Alpe di Melano. Nebst Bemerkungen über Komplexchromosomen. Chromosoma 16: 463-476. Seiler J, Schäffer K. 1960. Untersuchungen über die Entstehung der Parthenogenese bei Solenobia triquetrella F.R. (Lepidoptera, Psychidae) II. Analyse der diploid parthenogenetischen S. triquetrella. Verhalten, Aufzuchtresultate und Zytologie. Chromosoma 11: 29-102. 60 Simon JC, Delmotte F, Rispe C, Crease T. 2003. Phylogenetic relationships between parthenogens and their sexual relatives: the possible routes to parthenogenesis in animals. Biological Journal of the Linnean Society 79 151-163. Simon JC, Rispe C, Sunnucks P. 2002. Ecology and evolution of sex in aphids. Trends in Ecology and Evolution 17: 34-39. Smith FF. 1932. Biology and control of the black vine weevil. United States Department of Agriculture technical bulletin 325. Soltis DE, Soltis PS. 1993. Molecular data and the dynamic nature of polyploidy. Critical Reviews in Plant Science 12: 243-273. Soltis PS, Soltis DE. 2000. The role of genetic and genomic attributes in the success of polyploids. Proceedings of the National Academy of Sciences of the USA 97: 7051-7. Stebbins GL. 1950. Variation and evolution in plants. Columbia Univ. Press, New York. Stebbins GL. 1971. Chromosomal evolution in higher plants. Edward Arnold, London. Stebbins GL. 1985. Polyploidy, Hybridization, and the Invasion of New Habitats Annals of the Missouri Botanical Garden 72: 824-832. Stenberg P, Lundmark M. 2004. Distribution, mechanisms and evolutionary significance of clonality and polyploidy in weevils. Agricultural and Forest Entomology 6: 1-8. Stenberg P, Lundmark M, Knutelski S, Saura A. 2003a. Evolution of clonality and polyploidy in a weevil system. Molecular Biology and Evolution 20: 1626-1632. Stenberg P, Lundmark M, Saura A. 2003b. mlgsim: a program for detecting clones using a simulation approach. Molecular ecology notes 3: 329-331. Stenberg P, Terhivuo J, Lokki J, Saura A. 2000. Clone diversity in the polyploid weevil Otiorhynchus scaber. Hereditas 132: 137-142. Stoddart JA. 1983. A genotypic diversity measure. Journal of Heredity 74: 489-490. Stouthammer R, Breeuwer JA, Luck RF, Werren JH. 1993. Molecular identification of microorganisms associated with parthenogenesis. Nature 361: 66-68. Suomalainen E. 1940. Beiträge zur Zytologie der parthenogenetischen Insekten I. Coleoptera. Annales academiae scientiarum fennicae 7: 1-144. Suomalainen E. 1962. Significance of parthenogenesis in the evolution in insects. Annual Review of Entomology 7: 349-365. 61 Suomalainen E. 1969. Evolution in parthenogenetic Curculionidae. Evolutionary Biology 3: 261-296. Suomalainen E, Saura A. 1973. Genetic polymorphism and evolution in parthenogenetic animals. I. Polyploid Curculionidae. Genetics 74: 489-508. Suomalainen E, Saura A, Lokki J. 1987. Cytology and evolution in parthenogenesis. CRC Press, Boca Raton. Székessy W. 1937. Über Parthenogenese bei Koleopteren. Biologia generalis 12: 577-590. Turgeon J, Hebert PDN. 1994. Evolutionary interactions between sexual and all-female taxa of Cyprinotus (Ostracoda: Cyprinidae). Evolution 48: 1855-1865. Van Dijk PJ, Bakx-Schotman T. 1997. Chloroplast DNA phylogeography and cytotype geography in autopolyploid Plantago media. Molecular Ecology 6: 345-352. van Valen L. 1973. A New Evolutionary Law. Evolutionary Theory 1: 130. Vandel A. 1928. La parthénogenèse géographique. Contribution à'letude biologique et cytologique de la parthénogenèse naturelle. Bulletin biologique de France et de Belgique 62: 164-281. Vandel A. 1940. La parthénogenèse géographique.IV. Polyploidie et distribution géographique. Bulletin biologique de France et de Belgique 74: 94-100. Wanger RE, Negley FB. 1976. The genus Otiorhynchus in America north of Mexico (Coleoptera: Curculionidae). Proceedings of the Entomological Society of Washington 78: 240-262. Weeks AR, Marec F, Breeuwer JA. 2001. A mite species that consists entirely of haploid females. Science 292: 2479-2482. Weeks AR, Reynolds KT, Hoffman AA. 2002. Wolbachia dynamics and host effect: what has (and has not) been demonstrated. Trends in Ecology and Evolution 17: 257-262. Weismann A. 1889. The significance of sexual reproduction in the theory of natural selection. In: E. B. Poulton, Schauonland S and Shipley AE, eds. Essays upon heredity and kindred biological problems. Oxford: Clarendon Press. 251-332 Werren JH. 1997. Biology of Wolbachia. Annual Review of Entomology 42: 587-609. West-Eberhard MJ. 2005. The maintenance of sex as a developmental trap due to sexual selection. Quarterly Review of Biology 80: 47-53. 62 Wetherington JD, Kotora KE, Vrijenhoek RC. 1987. A test of the spontaneous heterosis hypothesis for unisexual vertebrates. Evolution 41: 721-731. White MJD. 1970. Heterozygosity and genetic polymorphism in parthenogenetic animals. In: Hecht MK and Steere WC, eds. Essays in evolution and genetics in honor of Theodosius Dobzhansky. New York: North Holland. 237-262. White MJD. 1973. Animal cytology and evolution. Cambridge University Press, Cambridge, England. White MJD. 1980. Meiotic mechanims in a parthenogenetic grasshopper species and its hybrids with related bisexual species. Genetica 54: 52-53. Viktorov AG. 1997. Diversity of polyploid races in the family Lumbricidae. Soil Biology and Biochemistry 29: 217-221. Williams GC. 1975. Sex and Evolution. Princeton University Press, Princeton. Vrijenhoek RC. 1979. Factors affecting clonal diversity and coexistance. American Zoologist 19: 787-797. Vrijenhoek RC. 1984. Ecological differentiation among clones: the frozen niche variation model. In: Wöhrmann K and Loeschcke V, eds. Population Biology and Evolution. New York: Springer-Verlag. 217-231. Yamane K, Yasui Y, Ohnishi O. 2003. Intraspecific CPDNA variations of diploid and tetraploid perennial buckwheat, Fagopyrum cymosum (Polygonaceae). American Journal of Botany 90: 339-346. Zhang L, Lefcort H. 1991. The effects on ploidy level on the thermal distributions of brine shrimp Artemia parthenogenetica and its ecological implications. Heredity 66: 445-452. 63