Genomic signatures of ancient asexual lineages

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Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2003? 2003

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Original Article

ANCIENT ASEXUAL GENOMESB. B. NORMARK

ET AL.

Biological Journal of the Linnean Society, 2003, 79, 69–84.

Intraclonal genetic variation: ecological and evolutionary aspects.

Edited by H. D. Loxdale FLS, FRES and G. Lushai FRES

Genomic signatures of ancient asexual lineages

BENJAMIN B. NORMARK 1 *, OLIVIA P. JUDSON 2 and NANCY A. MORAN 3

1

Department of Entomology, University of Massachusetts, Amherst, MA 01003, USA

2

Department of Biological Sciences, Imperial College, Exhibition Road, London, UK

3

Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA

Ancient asexuals – organisms that have lived without sex for millions of years – offer unique opportunities for discriminating

among the various theories of the maintenance of sex. The last few years have seen molecular studies

of a number of putative ancient asexual lineages, including bdelloid rotifers, Darwinulid ostracods, and mycorrhizal

fungi. To help make sense of the diverse findings of such studies, we present a review and classification of the predicted

effects of loss of sex on the eukaryotic genome. These include: (1) direct effects on the genetic structure of individuals

and populations; (2) direct effects on the mutation rate due to the loss of the sexual phase; (3) decay of genes

specific to sex and recombination; (4) effects of the cessation of sexual selection; (5) dis-adaptation due to the reduced

efficiency of selection; and (6) adaptations to asexuality. We discuss the utility of the various predictions for detecting

ancient asexuality, for testing hypotheses of the reversibility of a transition to asexuality, and for discriminating

between theories of sex. In addition, we review the current status of putative ancient asexuals. © 2003 The Linnean

Society of London. Biological Journal of the Linnean Society 2003, 79, 69–84.

ADDITIONAL KEYWORDS: adaptation – bdelloid rotifers – Darwinulid ostracods – eukaryotic genome –

molecular studies – mycorrhizal fungi – selection – sex.

INTRODUCTION

Ancient asexuals – eukaryotes that have lived without

sex for millions of years – pose a problem for evolutionary

theory. Asexuality is supposed to be a ticket to

a swift extinction (Maynard Smith, 1978); ancient

asexuals are predicted not to exist. Therefore, the persistence

of even a few ancient asexual groups is surprising,

and understanding how such organisms cope

without sex can potentially help us discriminate

between the various theories as to why sex is usually

necessary.

When we last reviewed this subject (Judson &

Normark, 1996), all claims of ancient asexuality

rested on negative evidence, such as the apparent

absence of males, and were thus easy to view with

scepticism (Hurst, Hamilton & Ladle, 1992; Little &

Hebert, 1996; Schön, Martens & Rossi, 1996). However,

the loss of sex is expected to have numerous

*Corresponding author. E-mail: bnormark@ent.umass.edu

genetic consequences. The detection of these consequences

would provide far more compelling evidence

that no sex has occurred for many generations – and

happily, the extraordinary recent technological

improvements in molecular genetics means that these

consequences are at last becoming detectable. As a

result, interest in putative ancient asexuals has

surged. Molecular evidence consistent with the claim

of the bdelloid rotifers to be bona fide ancient asexuals

was reported in 2000 (Mark Welch & Meselson, 2000),

and a vigorous empirical inquiry into bdelloid

evolutionary genetics is now underway (DB Mark

Welch & Meselson, 1998a; JL Mark Welch &

Meselson, 1998b; Arkhipova & Meselson, 2000; Mark

Welch & Meselson, 2001a,b). Empirical research programs

have also been launched to study ancient asexuality

in the Darwinulid ostracods (Butlin, Schön &

Martens, 1998; Rossetti & Martens, 1998, 1999;

Schön & Martens, 1998; Schön et al., 1998), the clam

genus Lasaea (Ó Foighil & Smith, 1995, 1996; Park &

Ó Foighil, 2000; Taylor & Ó Foighil, 2000), and the

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70 B. B. NORMARK ET AL.

vesicular arbuscular mycorrhizal fungi (Hijri et al.,

1999; Hosny et al., 1999; Kuhn, Hijri & Sanders, 2001;

Vandenkoornhuyse, Leyval & Bonnin, 2001); and the

pace of discoveries relevant to ancient asexuality in

other groups has quickened (Chaplin & Hebert, 1997;

Castagnone-Sereno et al., 1998; Sandoval, Carmean &

Crespi, 1998; Normark, 1999).

In this paper, we present a classification of predictions

of changes to genome structure and genome evolution

in ancient asexual lineages. We discuss the

utility of the various predictions for the purposes of (1)

corroborating cases of ancient asexuality, and (2) discriminating

between theories of the adaptive significance

of sexuality. We present an updated and revised

list of putative ancient asexual clades (for our original

list, see Judson & Normark, 1996) and we discuss the

prospects for corroboration of further cases and how

current evidence bears on the adaptive significance of

sexuality.

A FEW SEXY DEFINITIONS

By ‘sex’, we mean eukaryotic sex: meiosis followed by

the fusion of meiotic products from different individuals.

Asexuality refers to any reproductive process

that does not involve sex. Individuals may reproduce

through apomixis, that is by producing eggs through

mitosis. Individuals may reproduce vegetatively –

developing from multicellular propagules, for example,

by fission or budding. And individuals may

reproduce through automixis, producing eggs by first

undergoing meiosis and then re-fusing the meiotic

products, or by doubling the number of chromosomes

and then going through meiosis. Note that these different

types of asexuality have different genetic consequences.

Apomixis and vegetative reproduction give

rise to clones, individuals who inherit their single parent’s

genome in unrecombined (though slightly

mutated) form. Any heterozygosity present in the parent

is present in the offspring. The genetic outcome of

automixis depends on which meiotic products fuse;

automixis may be exactly equivalent to apomixis, it

may produce a situation where individuals become

completely homozygous, or it may produce complex

intermediate situations. For detailed discussion, see

Mogie (1986) and Suomalainen, Saura & Lokki (1987).

Note that when hermaphrodites self-fertilize, they

are engaging in what amounts to a special case of

automixis: the meiotic products are simply differentiated

into gametes before re-fusing. However, selffertilization

is much less likely to be a permanent condition

– self-fertile individuals usually retain a capacity

for outcrossing, and many self-fertile organisms

have sex at least occasionally. Unless otherwise specified,

in this paper ‘asexuality’ refers to all types of

asexuality except self-fertilization.

THE EFFECTS OF LOSING SEX:

AN OVERVIEW

Sex has profound effects on the genetic structures of

individuals and populations. In sexual populations,

novel combinations of genes are constantly formed

and destroyed. Indeed, any combination of genes in

the gene pool of an interbreeding population could

potentially arise. Alleles at different loci spread or go

extinct more or less independently of one another. And

as anyone who has tried to reconstruct a family tree

knows, the genealogical relationships between sexual

individuals take the form of a vast and complex

network.

In contrast, consider a strictly apomictic lineage.

When an apomict arises from its sexual ancestor, the

cycle of meiosis and fusion ceases, and from this point

forward only mitosis occurs. This leads to a simple

genealogical relationship: A begat B begat C begat D.

One parent, one grandparent, one great-grandparent.

The family tree of a group of apomictic lineages is not

a network, but a strictly branching tree. Mutation is

the only source of genetic novelty, and (assuming that

no recombination of any kind occurs) heterozygosity

will increase indefinitely. Moreover, there is no longer

any independent assortment of alleles: all loci show

complete linkage. Thus, every allele in the genome

should have the same history, a history of continual

branching and divergence.

Such a pattern of shared, congruent phylogenetic histories

is expected whenever entities are inherited

together without recombination. For example, we see

phylogenetic congruence between gene loci across species,

and between hosts and their obligate symbionts.

We even expect to see phylogenetic congruence between

species across geographical areas, at least to the extent

that the geographical history has been one of vicariance,

or the splitting of species ranges (Page, 1993).

By analogy, we refer to the expected pattern of allelegenealogy

congruence in asexual populations as

genomic panvicariance. In the case of automixis or obligate

selfing, genomic panvicariance between loci is

expected. In the special case of apomixis (and in the versions

of automixis that are equivalent), panvicariance

is expected to apply even to each pair of alleles that historically

were at the same locus in the sexual ancestor.

In addition to genomic panvicariance, asexuality is

expected to have strong effects upon particular classes

of genes. For example, since apomicts no longer carry

out meiosis, genes whose function is specific to meiosis

should start to degenerate or be co-opted to other functions,

and chromosomes should no longer be maintained

in pairs. In contrast, automicts should preserve

genes for meiosis, and chromosome pairing should be

conserved. In other respects, however, the two types of

asexuality should show the same patterns. Genes

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 69–84


ANCIENT ASEXUAL GENOMES 71

involved in male function (e.g. spermatogenesis) or in

female sexual behaviour (e.g. sex pheromones) should

degenerate or be co-opted to other functions. Genomic

parasites such as B chromosomes and transposable

elements can no longer bias meiosis to spread through

the population, and should gradually degenerate.

Finally, the loss of sex will have other, less predictable

effects. For example, the genome-wide substitution

rate may either increase or decrease – that will

depend on many different factors.

In Table 1, we have drawn together the various

effects that the loss of sex is predicted to have upon

the genome and split them into six categories. The categories

fall into two groups: direct (I–II) vs. indirect

(III–VI) effects of loss of sex. Direct effects are immediate

results of the loss of Mendelian genetics (I) or the

sexual cycle (II) per se. Indirect effects are the result of

different outcomes of selection on asexual as against

sexual lineages. The indirect effects can be further

subdivided into those resulting from the release from

selection of sex-related parts of the genome (III, IV)

and more general effects of the changed nature of

selection (V, VI). In the rest of this paper, we discuss

the various ways in which these predictions can be put

to use with respect to detecting ancient asexuality and

to shedding light on the adaptive significance of sex.

DETECTING ANCIENT ASEXUALITY

A population that is at least facultatively asexual is

easy to spot: virgins reproduce in solitary splendour,

and males appear to be absent. But it is harder to

demonstrate that observed asexuality is obligate, let

alone ancient. Thus, the first step in investigating an

asexual group is to determine that sex is truly absent,

and not simply rare or cryptic.

The easiest way to determine the absence of sex is to

test for phylogenetic congruence between loci within

populations (Table 1, I.A.1.i). Indeed, it has been

appreciated for many years that the lack of recombinants

between markers at different loci indicates a

clonal population structure (Burt et al., 1996; Smith &

Smith, 1998). But can this method also be used for

showing ancient asexuality? In our previous review,

we omitted discussion of this question as we assumed

the answer to be ‘No.’ We assumed this because a

clonal population structure is likely to completely supplant

a sexual population structure in a relatively

short time. Under a neutral model, the expected ‘coalescence

time’ – time since the most recent common

ancestor – is about 2N e generations, where N e is the

effective population size (Birky, 1996). The effective

population size is usually much smaller than the

actual census population size at any given time (Hartl,

1988) and the actual coalescence time for an asexual

population would probably be considerably less than

the neutral expectation 2N e , because all loci are completely

linked and any directional selection on any site

will tend to depress coalescence time. However, for

very tiny and abundant organisms, it is conceivable

that N e may be very high, especially in a metapopulation

with only limited gene flow between subpopulations,

and 2N e generations might be millions of years.

Even so, there is another problem with relying on

phylogenetic congruence between loci as an indicator

of ancient asexuality: other processes can also produce

phylogenetic congruence between loci. Loci sampled

from single exemplars of several non-interbreeding

species will typically show a high degree of phylogenetic

congruence. Thus, although excellent for confirming

current abstinence, the multilocus congruence

method is most likely to be useful in corroborating

ancient asexuality when it is used in conjunction with

other methods.

So what other methods are there? For apomicts, the

most powerful is to test for panvicariance at the allelic

level (Table 1, I.A.2). That is, to show that alleles at a

single locus (or more precisely, at what was a single

locus in the ancestral sexual genome) have diverged

prodigiously (Birky, 1996). Exactly this pattern of

divergence has recently been found for the bdelloid

rotifers (Mark Welch & Meselson, 2000) and in an

unusual variation on this theme, Hijri and colleagues

have found extreme heterozygosity among nuclei in

the multinucleate arbuscular mycorrhizal (AM) fungi

(Hijri et al., 1999; Hosny et al., 1999; Kuhn, Hijri &

Sanders, 2001).

However, failure to detect allelic panvicariance in a

genome does not necessarily mean that the organism

in question is not an ancient asexual. Serious departures

from allelic congruence may occur even under a

regime of strict asexuality (Birky, 1996). For example,

mitotic recombination may homogenize sequences.

Segments of DNA may be independently duplicated or

deleted. New segments of DNA may arrive through

the horizontal transfer of genes through non-meiotic

processes such as viral infection. In practice, therefore,

allelic panvicariance may often be extremely difficult

to show, and the technique may turn out to be of

disappointingly limited utility.

Moreover, although allelic panvicariance is strong

evidence for ancient asexuality, it is not conclusive.

Take the bdelloid rotifers again. Considering only the

patterns of divergence observed by DB Mark Welch

and Meselson, an extreme sceptic could invent alternative

explanations for the results. In principle, for

example, the bdelloids could be sexual: the observed

patterns might be due to an ancient genome duplication

followed by the maintenance of such high levels of

homozygosity that the allelic copies are indistinguishable.

An even more imaginative sceptic might argue

that the observed pattern of divergence is equally con-

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 69–84


72 B. B. NORMARK ET AL.

Table 1. A classification of predicted long-term genomic effects on genomes of the loss of sex and recombination

I. Direct effects on genetic structure of individuals and populations

A. Genomic panvicariance

1. Internuclear divergence

(i) Congruent divergence of many loci across organismal lineages within populations (Dykhuizen et al., 1993; Burt

et al., 1996)

(ii) Divergence of nuclear lineages within multinucleate organismal (fungal) lineages (Kuhn, Hijri & Sanders, 2001)

2. Intra-nuclear divergence: congruent divergence of alleles at one locus across species (Birky, 1996; Mark Welch &

Meselson, 2000)

B. Aneuploidy

1. Of entire karyotype: structural heterozygosity (JL Mark Welch & Meselson, 1998b; Blackman, Spence & Normark,

2000)

2. Of chromosome regions: e.g. loss or gain of ‘allelic’ tandem arrays (Blackman & Spence, 1996; Blackman et al. 2000)

3. Of individual ‘loci:’ loss (Little & Hebert, 1996) or gain of ‘alleles’ (Mark Welch & Meselson, 2000)

C. Disconcerted evolution: sequence divergence between copies within a tandem array (Fuertes Aguilar, Rosellò & Nieto

Feliner, 1999; Gandolfi et al., 2001)

II. Direct mutation-rate effect of loss of sexual phase (change in number of cell divisions per year)

A. Mutation rate (and substitution rate) decrease due to loss of mutagenic spermatogenesis (Redfield, 1994)

B. Mutation rate (and substitution rate) increase due to loss of dormant sexual phase (Normark & Moran, 2000)

C. Mutation rate (and substitution rate) decrease due to loss of mutations resulting from recombination

III. Decay of sex- and recombination-specific genes: pseudogene-like behaviour, resulting from release from selective

constraint. Genes potentially affected include those involved in shaping male genitalia, spermatozoa, seminal fluid

proteins, other male secondary sexual characters, copulatory behaviour (Carson et al., 1982), courtship behaviour,

pheromones, and (in apomicts) meiosis. Similar phenomena will affect transposable elements (Arkhipova & Meselson,

2000)

A. Speed-up of substitution rate

B. Deletions, insertions or complete loss

IV. Cessation of sexual selection: slowdown of substitution rate in genes that were subject to sex-related ‘arms races’

(sexual selection, interlocus contest evolution, maternal-fetal conflict) in sexual ancestors but that are still functional

in derived asexuals. Genes potentially affected include those involved in shaping female reproductive tract, egg chorion,

female reproductive behaviour

V. Dis-adaptation: increase of (deleterious) substitution rate due to reduced efficiency of selection (Moran, 1996;

Charlesworth & Charlesworth, 1997; Normark & Moran, 2000)

A. Due to decreased effective population size

B. Due to Muller’s ratchet in particular

VI. Adaptations to asexuality

A. Adaptations to prevent accumulation of deleterious mutations

1. Decrease of mutation rate

(i) Due to loss of transposable element activity (Arkhipova & Meselson, 2000; Hickey, 1982)

(ii) Due to enhanced DNA repair (Schön & Martens, 1998)

2. Innovations that increase mutational severity (Lynch et al., 1993)

(i) Decrease of gene copy number

(a) Decrease of ploidy

(b) Loss of duplicate loci

(ii) Loss of repair processes

(a) Loss of genes for proofreading

(b) Loss of break-repair enzymes

3. Innovations that decrease mutational severity; e.g. amplification of chaperonins (Moran, 1996; Normark & Moran,

2000)

B. Adaptations to resist parasites; e.g. speed-up of substitution rate of disease-resistance genes through adaptive locusspecific

loss of DNA repair

C. Co-optation of former sex- and recombination- specific genes to new function

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 69–84


ANCIENT ASEXUAL GENOMES 73

sistent with the bdelloids being recently asexual but

haploid. Such a lifestyle is possible: Weeks and colleagues

(Weeks, Marec & Breeuwer, 2001) recently

discovered the first species of strictly haploid female

metazoans, Brevipalpus phoenicis (Geijskes), a haploid

parthenogenetic mite. These mites appear to be

genetic haploid males that have been feminized by an

endosymbiotic bacterium. Like the mites, the bdelloids’

closest known sexual relatives, the monogonont

rotifers, are haplo-diploid. Thus, consider the following

scenario: (1) origin of haplodiploidy in the

ancestor of Monogononta + Bdelloidea; (2) divergence

of monogonont and bdelloid lineages; (3) genome doubling

in Bdelloidea (tetraploid females, diploid males);

(4) infection of a bdelloid by bacteria causing feminization

of genetic males; (5) spread of bacteria between

bdelloid species to infect most or all species; (6) loss of

male-determining genes from some or all bdelloid species;

and (7) loss of bacteria without reversion to sex.

In this scenario, the bdelloids were regularly sexual

until steps (4) and (5), and these steps can be postulated

to have occurred quite recently. Of course, this

scenario is rather implausible – it assumes steps (6)

and (7) must both have happened many times – but

it is nonetheless consistent with DB Mark Welch

and Meselson’s results. The allele divergences they

detected would date back to step (3).

More credible objections can be levelled at the congruence

found among nuclei in the VAM fungi. Consider

an AM fungus each of whose spores contain 1000

nuclei. If reproduction occurs through spores at any

appreciable rate, then the long-term effective population

size will not be much greater (and may be considerably

less) than this bottleneck size of 1000. Under

neutrality, the expected time to coalescence of all

nuclear lineages is 2N e , or about 2000 generations or,

in this context, 2000 nuclear replications – a rapid loss

of internuclear diversity compared to any reasonable

expectation of a per-locus mutation rate. Thus a priori,

the per-locus intraorganismal, internuclear divergence

is expected to be close to zero – and yet, the

observed internuclear divergence is very high (Kuhn,

Hijri & Sanders, 2001). How can this be explained? As

Sanders and colleagues acknowledge, one possibility is

that nuclei from hyphal networks derived from different

spores are being exchanged, a process that would

be equivalent to meiotic sex in many of its effects. A

second possibility is that the different nuclear lineages

have acquired complementary deleterious mutations

(Force et al., 1999): if different nuclei contain different

and complementary combinations of functional and

non-functional genes, loss of any one of the nuclear lineages

may be lethal. Internuclear diversity would then

be preserved by vigorous natural selection against

spores that did not contain a complete complement of

nuclei.

Fortunately for ancient-asexual sleuths, there are

other tools in the ancient-asexual-detection toolkit.

First, there’s karyotype structure (Table 1, I.B). The

discovery that apomicts maintain chromosomes in

homologous pairs would strongly discredit a claim of

ancient asexuality, for example. Conversely, showing

that genomes do not contain more than one copy of any

given divergent allele sequence would remove doubts

that observed allele congruences are actually the

result of a genome duplication followed by the maintenance

of high levels of homozygosity, and thus would

further bolster a claim of ancient asexuality. Indeed,

in the case of the bdelloids, in situ hybridization has

shown that each allelic sequence is found only once in

each genome (JL Mark Welch and Matthew Meselson,

pers. comm.). Note, however, that although a strongly

aneuploid or structurally heterozygous karyotype is a

good indicator of asexuality, it is not, by itself, a good

indicator of ancient asexuality. Recent work in aphids

shows that once sex is lost, genome structure can

become irregular extremely quickly (Normark, 1999;

Wilson, Sunnucks & Hales, 1999; Spence & Blackman,

2000). Similarly, one of the two homologous copies of

the ribosomal DNA array is typically lost shortly after

aphids abandon the sexual phase of their life cycle

(Blackman & Spence, 1996).

Another way to investigate claims of ancient asexuality

is to look for evidence of the decay of genes that

have an exclusively sexual function (category III).

Crippling deletions in genes known to be crucial to

meiosis and spermatogenesis would suggest that the

organisms bearing those deletions are asexual; the

discovery that deletions are shared with a related species

would suggest the common ancestor of the two

species was also asexual. The same goes for selfish

genetic elements such as retrotransposons, which are

expected to degenerate in the absence of meiosis

(Hickey, 1982) and, indeed, the bdelloid rotifers lack

two major classes of retrotransposon that are found in

all other metazoans that have so far been investigated

(including the bdelloids’ closest known relatives, the

monogononts; Arkhipova & Meselson, 2000). Otherwise,

though, the data for genetic decay are limited –

for now.

As more and more genes involved in meiosis and

spermatogenesis are identified, the search for the

presence or absence of many of these genes will start

to become feasible. Yet this approach, too, will have

pitfalls. If a group of organisms has been apomictic for

long enough the genes for meiosis may have disappeared

completely; the sceptical may attribute the

failure to find particular genes to investigator incompetence.

Worse still, if the genes are present and do

not show the expected decay, it may be extremely difficult

to demonstrate that the reason they have

remained intact is not due to the organism having sex

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74 B. B. NORMARK ET AL.

but rather to the genes’ involvement in additional

functions, or to their having been co-opted to new functions.

Involvement or co-option are both strong possibilities.

Many genes involved in meiosis may also be

involved in DNA repair, for example, and although the

putatively ancient asexual protozoan Giardia lamblia

has lost one class of retrotransposon, it does have two

classes of active retrotransposons; both of these are

active only in telomeric regions of chromosomes and

may well have become involved in telomere extension

(Arkhipova & Morrison, 2001).

In short, the corroboration of claims of ancient asexuality

remains extremely difficult. Undoubtedly, the

best hope is to combine several different approaches.

For example, taken together, the facts that the bdelloid

rotifers not only (1) show allelic panvicariance,

but also (2) do not have more than one copy of each

divergent sequence, (3) lack two major classes of retrotransposon,

and (4) do not have homologous chromosomes,

give overwhelming support to the argument

that the bdelloids are bona fide ancient asexuals.

What of the status of other groups on our list of candidate

ancient asexuals (Table 2)? To date, no other

group has been investigated as thoroughly as the bdelloids;

however, intriguing results from several other

taxa are starting to come in. For example, the Lasaea

clams show huge levels of heterozygosity and polyploidy

(Ó Foighil & Smith, 1995, 1996; Park & Ó

Foighil, 2000; Taylor & Ó Foighil, 2000); they may well

turn out to show allelic panvicariance. Against that,

these clams are pseudogamous selfing hermaphrodites

– they need to produce sperm in order to initiate

embryonic development. Although the male genome is

thrown out, such a situation may allow for occasional

outcrossing or the incorporation of additional nuclei. If

this were the case, then the observed heterozygosity

would simply be the consequence of multiple, independent

incorporations of male DNA.

Although there is no evidence that the oribatid

mites are, after all, having sex, investigation of their

claim to ancient asexuality has been complicated by

the fact that allelic panvicariance has already been

ruled out: electrophoretic data shows appreciable

levels of homozygosity (Palmer & Norton, 1992). But

since many of these mites probably have enormous

population sizes, this is a group where ancient asexuality

may potentially be shown through the study of

clonal population structures in tandem with the study

of the decay of genes involved in sex.

The status of the Darwinulid ostracods is similarly

uncertain. Gandolfi et al. (2001) have shown that the

Darwinulids have high levels of heterozygosity in the

repeats of the 18S ribosomal RNA units, as predicted.

However, since little is known about the average levels

of divergence shown by sexuals, the significance of this

finding is not yet clear.

ASEXUALS AND THE ADAPTIVE

SIGNIFICANCE OF SEX

A brief glance at the taxonomic distribution of asexuals

gives the immediate impression that although

asexuality often arises, it rarely persists for long.

Indeed, as has often been remarked, the bdelloid rotifers

are highly unusual in being a whole class in which

sex is unknown. The rapid extinction of most asexuals

is the chief reason that evolutionary biologists believe

sex to be essential. But it remains possible that the

rapid extinction of asexual groups is an illusion. As

Williams (1975) observed, the taxonomic distribution

could be equally well explained if asexuals often revert

to sex.

If this interpretation were correct, the nature of the

problem would be quite different. Asexuality would

not be a ticket to extinction. Indeed, many organisms

could have bouts of asexuality in their ancestry and

we would have no way of knowing about it. The question

would not be why is asexuality a dead end, but

why is sex so hard to quit? Over the past 25 years, this

alternative interpretation of the taxonomic distribution

has largely been ignored. However, we think that

this is an important matter to resolve, and that it can

now be resolved empirically.

REVERSING ASEXUALITY

The key to determining whether or not asexuals typically

revert to sex is to quantify the speed with which

asexuality becomes irreversible. Predictions from category

III of our Table 1 can be used to investigate this

question. It seems likely a priori that the different

types of asexuality will have different probabilities of

reverting. Since automicts maintain genes for meiosis,

resuming outcrossing should be easier for automicts

than for apomicts. In populations of obligately selffertile

hermaphrodites, outcrossing may easily resume

if males reappear. For example, in the mangrove fish,

Rivulus marmoratus Poey, all investigated populations

are obligately self-fertile hermaphrodites except

those of Honduras and Belize, where males comprise

2% and 25% of the populations, respectively (Taylor,

Fisher & Turner, 2001); phylogenetic analysis shows

that the populations containing males are derived

rather than ancestral (Weibel, Dowling & Turner,

1999).

In contrast, if the genomes of apomicts contain large

deletions from genes typically used for meiosis – or,

more generally, if genes involved in spermatogenesis

and other aspects of male reproductive function, or

genes involved in female sexual behaviour – have obviously

degenerated, a return to sex is likely to be difficult,

perhaps impossible. Furthermore, when the

origin of asexuality is known, the speed with which

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ANCIENT ASEXUAL GENOMES 75

Table 2. Candidate ancient asexual clades of eukaryotes. Under ‘Estimated age’, estimates reported from the literature are given in millions of years (Myr). The

basis of the age estimate is indicated as follows: m = molecular, f = fossil, g = geography; c = crown clade age (i.e. based on sequence divergences or fossils within

the putative ancient asexual clade itself); s = stem clade age (i.e. based on divergence from closest sexual relative)

Taxonomic

affiliation Clade

Estimated

age

Myr Basis

No. spp. Reproduction Notes Refs

(A) DECISIVELY CORROBORATED ANCIENT ASEXUAL

Rotifera Bdelloidea >30 f, c 363 Apomictic See text Mark Welch & Meselson

(2000); Poinar & Ricci (1992)

(B) CANDIDATE ANCIENT ASEXUALS

Eukaryota

(‘Protista’)

Diplomonadida ? – – Mitotic Arkhipova & Morrison (2001);

Dacks & Roger (1999)

Eukaryota

(‘Protista’)

Pteridophyta:

Vittariaceae

Pteridophyta:

Hymenophllaceae

Agaricales:

Lepiotaceae

Agaricales:

Tricholomataceae

Entamoebida ? – – Mitotic Dacks & Roger (1999)

Vittaria sp. 10 g, s 1 Vegetative

(gemmae)

Trichomanes sp. 10 g, s 1 Vegetative

(gemmae)

fungal cultivar of

higher attine ants

fungal cultivar of

Apterostigma ants

25 f, c ? Vegetative (ant

culture)

? – ? Vegetative (ant

culture)

Mollusca NE Pacific Lasaea 3 m,c 1? Pseudogamous

selfing

hermaphrodite

Mollusca Lasaea

SAF-NZL-KERG

? – 1? Pseudogamous

selfing

hermaphrodite

Mollusca Lasaea JPN 1–3 ? – 1? Pseudogamous

selfing

hermaphrodite

Mollusca Lasaea IR2 ? – 1? Pseudogamous

Mollusca Lasaea Flor-

LRAUSDD-JPN4

Mollusca Lasaea IR1,IR3,

FRMED

selfing

hermaphrodite

6 m,s 1? Pseudogamous

selfing

hermaphrodite

? – 1? Pseudogamous

selfing

hermaphrodite

Farrar (1990)

Farrar (1990)

Chapela et al. (1994); Hinkle et al.

(1994); Mueller et al. (1998)

Chapela et al. (1994); Hinkle et al.

(1994); Mueller et al. (1998)

Ó Foighil & Smith (1995);

Ó Foighil & Smith (1996);

Taylor & Ó Foighil (2000)

Taylor & Ó Foighil (2000)

Taylor & Ó Foighil (2000)

Taylor & Ó Foighil (2000)

Ó Foighil & Smith (1995);

Ó Foighil & Smith (1996);

Taylor & Ó Foighil (2000)

Taylor & Ó Foighil (2000)

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76 B. B. NORMARK ET AL.

Table 2. Continued

Taxonomic

affiliation Clade

Estimated

age

Myr Basis

No. spp. Reproduction Notes Refs

Mollusca:

Gastropoda

Arthropoda:

Ostracoda

Arthropoda:

Ostracoda

Campeloma

parthenum E.

1.3 m, s 140 ? Norton et al. (1993); Balogh &

Balogh (1987)

Arthropoda: Acari Brachychthoniidae ? – >100 ? Norton et al. (1993); R. A. Norton,

in litt.

Arthropoda: Acari Trhypochthoniidae ? – 24 ? Norton et al. (1993)

Arthropoda: Acari Malaconothridae ? – 20 ? Norton et al. (1993)

Arthropoda: Acari Camisiidae ? – 32 ? Norton et al. (1993)

Arthropoda: Acari Nanhermannidae ? – 12 ? Norton et al. (1993)

Arthropoda: Acari Nothrus ? – 24 ? Norton et al. (1993)

Arthropoda:

Phasmatodea

Timema douglasi 1.5 m, s 1 ? Sandoval et al. (1998)

Arthropoda:

Phasmatodea

Arthropoda:

Phasmatodea

Timema genevieve 1.5 m, s 1 ? Sandoval et al. (1998)

Timema tahoe 1.5 m, s 1 ? Sandoval et al. (1998)

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ANCIENT ASEXUAL GENOMES 77

Taxonomic

affiliation Clade

Estimated

age

Myr Basis

No. spp. Reproduction Notes Refs

(C) COMPLICATED CASES: EVIDENCE AGAINST STRICT CLONALITY

Zygomycota: Zygomycotina

Glomales 400 f, c >100 Azygospores Exchange of nuclei

among multinuclear

fungi

Ascomycota:

Ophiostomales

Ascomycota:

Ophiostomales

Ascomycota:

Ophiostomales

Nematoda:

Heteroderidae

Arthropoda:

Ostracoda

Arthropoda:

Coleoptera

Chordata:

Lissamphibia

Arthropoda:

Phasmatodea

‘Ambrosia’ fungus of

platypodine and

corthyline beetles

‘Ambrosia’ fungus of

xyleborine and

xyloterine beetles

‘Ambrosia’ fungus of

ipine beetles

60 m, s ? Incongruence of

fungal/beetle phylogeny

35 m, s ? Incongruence of

fungal/beetle phylogeny

21 m, s ? Strict vertical transmission

by host unlikely

Meloidogyne spp. ? – 4 Apomictic Some inconguence of

nuclear and mtDNA

phylogeny

Eucypris virens 4 m, c 26 Apomictic Some inconguence of

nuclear and mtDNA

phylogeny

Aramigus tessellatus 2 m, c


78 B. B. NORMARK ET AL.

such degeneration appears can be measured. For

example, Carson, Chang & Lyttle (1982), working

with a parthenogenetic strain of Drosophila mercatorum

Patterson & Wheeler, found that female sexual

behaviour decayed almost completely within 20 years;

if such an effect could be shown to be due to loss of

gene function, this would constitute powerful evidence

that transitions to asexuality can quickly become

irreversible.

In hymenopteran parasitoids that are thelytokous

due to the effects of infection by Wolbachia pipientis

Hertig, curing the infection with antibiotics can result

in males and females that fail in essential sexual

functions including sperm viability, male ejaculation

ability, and female willingness to mate (Gottlieb &

Zchori-Fein, 2001). Although these data are sketchy,

they suggest that such transitions to asexuality may

indeed be irreversible: infected asexual lineages

swiftly accumulate multiple mutations that destroy

aspects of the sexual phenotype.

THEORIES OF SEX

Assuming that the observed taxonomic distribution

really does reflect the rapid extinction of asexuals, the

question is, why is sex necessary, and how do any

ancient asexuals manage to persist? Furthermore,

each theory of sex must also be able to explain the geographical

distribution of obligate asexuality: that

asexuality is much more common in freshwater systems

than in the ocean, that it is more often found at

high latitudes and high altitudes, and so on (Bell,

1982). After all, the null hypothesis must be that asexuality

appears in populations with equal frequency

regardless of the environment, but that it can start to

flourish in some environments and not in others.

More than 20 theories have been put forward to

explain sex (Kondrashov, 1993); here we discuss the

three that we consider to be the front-runners:

Muller’s ratchet, Kondrashov’s hatchet, and the Red

Queen.

MUTATIONAL THEORIES

Both Muller’s ratchet and Kondrashov’s hatchet propose

that the benefit of sex derives from the fact that

sex eliminates deleterious mutations. However, the

two theories differ completely with respect to the predictions

they make.

According to Kondrashov’s hatchet – also known as

the mutational deterministic hypothesis (Kondrashov,

1993) – sex is essential if the rate of deleterious mutations

per genome per generation (U) is greater than

one. If the deleterious mutation rate is higher than

that, asexuals should become extinct within a few generations

of their appearance, and ancient asexuality is

not a possibility. In order to account for ancient asexuality

under Kondrashov’s hatchet, it is necessary to

postulate that a few lineages must escape the hatchet

by having less than one deleterious mutation per

genome per generation (U < 1.0). Such a low deleterious

mutation rate may be due to a low mutation rate

(e.g. Table 1, II.A, II.C, VI.A.1), or to a low severity of

mutations (e.g. Table 1, VI.A.3). Thus, Kondrashov’s

hatchet predicts that the phylogenetic and ecological

distribution of asexuality should be correlated with

low per-genome mutation rates (small genomes, low

incidence of transposable elements, low radiation

exposure) or with relatively benign environments in

which mutational severity is reduced. A few ancient

asexual lineages are endosymbionts or cultivars of

other species (Law & Lewis, 1983; Judson & Normark,

1996), and as such may indeed experience reduced

mutational severity. However, the broad pattern of

ecological distribution of asexuality does not favour

the hatchet (Bell, 1982; Burt, 2000).

In contrast to Kondrashov’s hatchet, Muller’s

ratchet predicts that asexuals living in small populations

will be slowly driven extinct through the inexorable

accumulation of deleterious mutations. Muller’s

ratchet has been extensively explored theoretically

(e.g. Kondrashov, 1994; Butcher, 1995; Gordo, Navarro

& Charlesworth, 2002), and depending on the assumptions

made regarding epistasis, population size, the

backwards mutation rate and other variables, it is predicted

to eliminate asexuals rapidly, to eliminate them

slowly, or not to operate at all. However, there is

substantial evidence for mutation accumulation in

asexual microbes subject to genetic drift. Studies of

experimental evolution in RNA viruses have shown

that deleterious mutations accumulate in small populations

and that recombination can reduce mutation

frequencies and restore fitness in these viruses (Chao,

1990; Chao, Tran & Tran, 1997). Some eubacteria

have lost recombination as a result of long-term

evolution as vertically transmitted obligate symbionts

of insects (Moran, 1996; Spaulding & von Dohlen,

1998; Clark, Moran & Baumann, 1999). DNA

sequences from these bacteria lineages show elevated

rates of substitution concentrated at sites resulting in

amino acid replacements (non-synonymous sites).

This concentration is expected since replacement sites

are the ones at which mutations are likely to affect

fitness.

In addition to showing faster sequence evolution,

the genomes of long-term symbionts and pathogens

show the loss of genes that are non-essential but

that function to stabilize central cellular processes

(Andersson & Kurland, 1998; Moran & Wernegreen,

2000; Shigenobu et al., 2000). Mutations knocking out

such genes are deleterious but not always lethal, and

such mutations are more likely to spread to fixation

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 69–84


ANCIENT ASEXUAL GENOMES 79

under asexuality and small population size. Apparently

because of this loss of gene function by genetic

drift, genomes of obligate symbionts and pathogens

contain fewer functional genes than genomes of

related free-living lineages that retain recombination.

Asexual symbiont genomes so far characterized show

evidence of degradation and shrinkage (Shigenobu

et al., 2000; Clark et al., 2001). The lost genes may

include those that enable intra- and intergenomic

recombination. For example, the drecA pathway is

omitted from Buchnera, the long-term bacterial symbiont

of aphids; this is expected to eliminate most

homologous recombination, which has a sex-like role

in bacterial populations, allowing exchange of DNA

between homologous chromosomes of different organisms.

Thus the genomic signature of long-term

absence of recombination in Buchnera includes fast

polypeptide evolution, loss of genes, destabilized gene

products, and elimination of loci that underlie homologous

recombination. However, since symbionts such

as Buchnera also show other distinctive lifestyle

attributes, which lead to reduced effective population

sizes, it is presently difficult to tell whether these phenomena

are primarily due to the loss of recombination,

an adaptive response to being a symbiont (genome

shrinkage may allow faster replication, which may be

advantageous), or simply the result of increased

genetic drift.

Complicating matters further, the expectation of an

elevated rate of non-synonymous substitutions in

asexual lineages is based on the presumption that

these mutations are mildly deleterious to fitness.

However some of the observed mutations may be

compensatory. Clear evidence of compensatory mutations

is available for asexual symbionts in which

faster rates of substitution in ribosomal RNA genes

leads to mismatches in the paired stem structures

but also to changes in both paired nucleotides

(Lambert & Moran, 1998). For example, many G-C

pairs have been replaced by A-T pairs and these

replacements must have occurred as two substitutions

with the second one being compensatory and driven in

part by selection. Similarly, a portion of the amino

acid replacements in fast-evolving polypeptides is

probably compensatory, acting to stabilize the protein

structure in the face of de-stabilizing mutations fixed

previously.

Rampant amino acid substitutions are expected to

result in proteins with less stable secondary structure.

One apparent adaptation in bacteria that increases

the functionality of destabilized proteins is the overexpression

of chaperonins that refold polypeptides

having lost their secondary structure (Moran, 1996).

Normally these chaperonins are facultatively expressed

when the cell is stressed, as under heat-shock.

They interact with a wide range of proteins in an

energy-consuming process that re-establishes the

proper folded structure. A host of symbiotic bacteria

and pathogens show constitutive high expression of

groEL protein, which functions in the folding of a large

set of gene products. This over-expression may represent

a compensatory adaptation to accumulation of

slightly deleterious, destabilizing mutations throughout

the genome. Recent experimental studies have

shown that over-expression of groEL can restore fitness

levels in long-term lineages of Escherichia coli

that have accumulated deleterious mutations (Fares

et al., 2002). Chaperonins can also mask mutations in

eukaryotes (Carmichael et al., 2000), and chaperonin

over-expression would be a simple feature to assay in

eukaryote lineages suspected of ancient asexuality.

In principle, adaptive responses to mutation accumulation

could be either to increase the severity of

mutations, or to reduce the mutation rate (Table 1,

VI.A). However, since some mutations are inevitable, it

may be difficult to reduce the mutation rate below a certain

threshold without enormous extra cost. Certainly,

long-term bacterial symbionts do not appear to have an

adaptive decrease in mutation rate. In fact, calibrations

of silent substitutions against absolute time suggest

that the mutation rate is elevated in Buchnera

relative to its free-living, recombining relatives (Clark

et al., 1999); this might be expected as a result of loss

of repair genes. In bacteria, sex and repair (which

determines mutation rate) are aspects of the same processes

and so it is difficult to envision that loss of recombination

will be accompanied by more efficient repair.

For example, the mutation rate may increase as a

result of loss of recA, which can enable recombinational

repair processes as well as gene exchange.

In eukaryotes, a decreased mutation rate has been

claimed for the putative ancient asexual, Darwinula

stevensoni (Brady & Robertson) (Schön & Martens,

1998); however, the basis of this claim has been questioned

(Normark & Moran, 2000; Gandolfi et al.,

2001), and its significance is unclear. The only other

group that has been investigated, the bdelloid rotifers,

do not have a substitution rate that differs from that

of the monogononts (Mark Welch & Meselson, 2001b).

The similarity of the monogonont and bdelloid substitution

rates is remarkable, given the variety of processes

expected to alter substitution rates in asexuals

(Table 1, II, V, VI.A). One interpretation is that both

groups may be near the threshold of the lowest mutation

rate possible – thus the secret of the bdelloids’

success may be that they originated from an ancestor

with an unusually low mutation rate.

Muller’s ratchet can potentially explain why some

asexuals persist for an appreciable number of generations,

and why most asexuals fail to become ancient.

Note, however, that Muller’s ratchet is not generally

expected to apply in asexual populations with very

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80 B. B. NORMARK ET AL.

large populations; nor is there any obvious way that it

can explain the geographical incidence of asexuality.

THE RED QUEEN

The main alternative to the mutational theories of sex

is a theory known as the ‘Red Queen’ (Hamilton,

2001). In its most general form, the Red Queen states

that sex is an advantage in antagonistic coevolutionary

interactions, whether between predators and prey,

between competitors within a species, or between

hosts and their harmful parasites. Of these various

interactions, those between hosts and harmful parasites

are believed to be the most important in maintaining

sex, for at least two reasons. First, interactions

between hosts and parasites typically have a large

genetic component. As a result, individuals with rare

or novel genotypes are less likely to be susceptible to

infection. Second, parasites tend to be short-lived in

comparison to their hosts, and thus go through many

generations in the course of each host generation. As a

result, parasites can quickly evolve to infiltrate host

defences. In other words, genes that are good to have

today won’t be the genes that are good to have tomorrow,

and sex is an advantage precisely because it

breaks up existing gene combinations and generates

new ones.

By this logic, asexuals are doomed as follows. A genotype

that is initially rare, and thus resistant to parasite

attack, will rise in frequency. As it does so, it

presents a larger target to parasites, which can then

evolve to attack it: clones become the victims of their

own success. Moreover, because a clone’s genotype at

any given locus is likely to be identical to that of her

mother and grandmother, the target presented by a

given genotype is stable through time. Parasites that

are initially poor at infecting a given genotype can,

after a few host generations, become devastatingly

effective. (Note that the virulence of a parasite is

thought to depend chiefly upon its own opportunities

for transmission, and that there is no reason to suppose

that a given parasite will necessarily evolve to

become less dangerous over time.)

Recent experiences from human agriculture testify

to the power of parasites to ravage asexual populations.

In western farming, crops are generally planted

as monocultures with low genetic diversity, a procedure

that increases yield, but that notoriously leaves

crops vulnerable to disease. Once a disease takes hold,

it can sweep through the entire stand. The invention

of disease-resistant strains of crops is therefore a constant

preoccupation of plant breeders and biotechnology

companies, and new strains are regularly brought

onto the market. But, alas, any triumph over parasites

is always temporary: within a few seasons, a once

pest-resistant strain resists no more (Palumbi, 2001).

So how, then, can asexuals escape extinction? How is

ancient asexuality possible?

According to the Red Queen, the benefit of sex is

hypothesized to stem from the fact that sex generates

novel, rare, transient genotypes. Sex is not the only

way to acquire a rare genotype, however. Under most

conditions migration will be equivalent to sex, at least

in this respect. An asexual individual who migrates

from an area where its genotype is common to one

where its genotype is rare may, if it can migrate without

its harmful parasites, gain the Red Queen benefit

of sex without the costs (Ladle, Johnstone & Judson,

1993). Ladle and colleagues proposed that dormant

stages such as anhydrobiosis might be one way to

effect parasite-free dispersal, and certainly, intracellular

parasites such as Wolbachia are often lost during

dormant stages (M-J. Perrot-Minot, pers. comm.). Consistent

with this idea, those bdelloid rotifers that survive

anhydrobiosis are greatly rejuvenated (Ricci,

1987), although the reason for this, and any possible

connection to bdelloid parasite loads, is presently

obscure.

Less speculative evidence that parasite-free dispersal

can allow sex under the Red Queen comes from

the ancient asexual fungus cultivated by the higher

attine ants. The fungus is susceptible to a virulent disease

known as Escovopsis; an attack of Escovopsis can

wipe out an attine fungus garden and consequently

destroy the ant colony. In keeping with the view that

parasites can easily evolve to ravage a host that has

been asexual for many generations, Currie and colleagues

(Currie, Mueller & Malloch, 1999) found that

Escovopsis is more virulent on the ancient fungus cultivated

by the higher attines than it is on fungi domesticated

more recently. However, when a young queen

flies off to found a new colony, she carries with her cultivars

of the fungus, and a filamentous bacterium that

produces antibiotics specific to Escovopsis (Currie,

Scott et al., 1999). That the dispersal of fungus

between colonies is achieved in the absence of Escovopsis

has been corroborated by Currie, Mueller et al.

(1999), who have shown that Escovopsis is probably

only transmitted horizontally.

If the chief benefit from sex comes from having a

genotype that is unfamiliar to parasites, then having

a higher mutation rate in genes involved in host–

parasite interactions would be adaptive for asexuals

(Table 1, VI.B), and could potentially be enough to

allow them to escape extinction. Such an effect is presently

unknown. However, there are several mechanisms

that could, in principle, generate mutations in

particular parts of the genome. A gene’s position on

the chromosome may affect the rate at which it is copied

and therefore the number of mutations that occur;

genes involved in parasite resistance might be clustered

in high-mutation-rate regions of the genome.

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 69–84


ANCIENT ASEXUAL GENOMES 81

Alternatively, DNA repair of particular loci may be

prevented. Or, asexuals may increase their somatic

mutation rate when under parasite attack, an effect

that has already been shown to occur in the vertebrate

immune system (Sale et al., 2001). Different mechanisms

would be predicted to have different effects on

the observed substitution rate of genes involved in

parasite responses. If asexuals elevate their somatic

mutation rate, then the observed substitution rate of

genes involved in parasite resistance may not differ

from the substitution rates in other genes. However,

there is no evidence that such mechanisms have

evolved.

Unlike the mutational theories, the Red Queen

seems a good candidate to explain the geographical

incidence of asexuality. Note, however, that there is

presently little direct evidence in support of the Red

Queen, and that the general predictions of the theory

are frustratingly vague.

WHAT WE STILL NEED TO KNOW

Alas, most questions that are key to elucidating the

adaptive importance of sex remain unanswered. The

antithetical questions of the frequency of asexual origin

and the frequency of reversion to sex both need

serious attention. Mutation rates of more asexuals,

both ancient and modern, and of their close sexual

relations need to be measured. Almost nothing is

known about the parasites of any putative ancient

asexual, nor of the effects of aspects of the life cycle

such as dormancy on parasite load, a deficiency that

needs to be rectified. Genome organization, and the

variation of mutation rates within the genome need to

be measured. In sum, much work remains to be done.

ACKNOWLEDGEMENTS

Many thanks to Austin Burt and Jonathan Swire

for stimulating discussions, to Roy Norton for useful

criticism of our previous list of candidate ancient

asexuals, and to Hugh Loxdale and two anonymous

reviewers for many helpful comments that improved

the manuscript.

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