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-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
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