Maintenance of aphid clonal lineages: images of immortality?

Infection, Genetics and Evolution 3 (2003) 259–269

Maintenance of aphid clonal lineages: images of immortality?夽 Hugh D. Loxdale∗ , Gugs Lushai Plant & Invertebrate Ecology Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK Received 31 January 2003; received in revised form 18 June 2003; accepted 18 June 2003

Abstract Artificial cloning and ancient asexuals have impacted upon both scientific and lay thinking in applied and theoretical fields as diverse as medicine and evolution. Hence, this is an opportune time to promote debate and discussion on what maintains a clonal lineage. The genetic fidelity of a clone has been discussed in detail elsewhere [Genet. Res. 79 (2002) 1; Biol. J. Linnean Soc. 79 (2003) 3]. In this paper, we focus on the lineage integrity (=longevity), or physiological lifespan of a clone with respect to senesce in relation to factors controlling telomere functioning. Aspects of cell line research pertinent to eukaryotic clonal lineages are discussed and, in particular, we try to extrapolate aspects of this research and apply it to apomictic (=mitotic) aphid lineages to suggest how they may be maintained. Analogies are made between single cells and individual aphids that senescence through a generation, whilst the respective lineages persist for finite periods, unless that is, compensatory mechanisms have evolved allowing immortality in the one and ancient asexuality in the other. Such comparison may allow fresh insights into the mechanisms of clonal lineage maintenance and evolution. We hypothesise that: (1) the cause of extinction in eukaryotic clonal lineages is due to deleterious effects on key regions of the genome, the chromosomal telomere being one such site; (2) recombination acts as a common mechanism to reset telomere functioning, perhaps more fundamental than its utility to reduce genetic load and maintain adaptability; and (3) ancient lineages persist through time as a function of group-specific compensatory mechanisms that maintain telomere integrity. © 2003 Elsevier B.V. All rights reserved. Keywords: Clonal lineage; Asexual; Sexual recombination; Ancient asexuals; Telomere; Evolution; Ageing; Senescence

1. Introduction Aphid propagation, which mainly involves parthenogenesis, appears to show certain similarities with the propagation of somatic cells. As in whole sexual animals, cell lineages have a defined lifespan. Cells normally divide up to 15–60 times (=generations) in vitro leading to senescence and death (Hayflick, 1965; Rawes et al., 1997). In contrast, some clonal lineages of whole organisms could be ancient and potentially immortal (Normark et al., 2003). In this respect, such eukaryotic clonal lineages are analogous to cancer cells, i.e. they have mutations and are uncontrolled in terms of proliferation, and are not limited by a set number of replications as far as known. Some artificially reared aphid lineages (Hemiptera: Aphididae) have been kept in culture for around 50 years (∼1000 generations) (Powell and Hardie, 2000). 夽 Note from the Editor-in-Chief: This article does not deal directly with infectious diseases. However, since Infection, Genetics and Evolution aims at being a forum journal, I found this work relevant to our field, considering the importance of clonal evolution in many pathogenic microorganisms. ∗ Corresponding author. Tel.: +44-1582-763133; fax: +44-1582-760981. E-mail address: [email protected] (H.D. Loxdale).

1567-1348/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-1348(03)00091-1

Aphids are thus potentially a valuable and easily exploitable resource to understand how lineages persist over time. In general, aphid species reproduce asexually throughout the spring and summer months, only indulging in a period of sexual reproduction in the autumn when sexual forms mate and produce overwintering eggs (=holocyclic lifecycle, i.e. facultative asexual with annual sexual phase). Some species display lifecycle types which may continue to reproduce asexually even through the winter, i.e. are anholocyclic (obligate asexual), androcyclic (obligate asexual with some males) and intermediate (obligate asexuals with a few of both sexes; see Dixon, 1998 and Simon et al., 2002). A few species seem to be obligate asexuals, reproducing parthenogenetically all the year around, e.g. the shallot aphid, Myzus ascalonicus Doncaster and in which sexual forms have so far not been found (Blackman and Eastop, 2000). Asexual propagation in aphids is of the apomictic (=mitotic) type, and there is no strong evidence for any form of endomeiosis or internal chromosomal recombination (Blackman, 1979; Tomiuk and Wöhrmann, 1982; but see Mandrioli et al., 1999). Holocyclic asexual aphid lineages may produce ∼12–14 asexual generations per annum interspersed with the single annual sexual phase (Dixon, 1998), whereas obligate

260

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

asexual species like M. ascalonicus can probably produce up to 20 generations per year in warmer climes. The asexual phase involves a telescoping of generations with the simultaneous development of three generations, like a biological ‘Russian doll’, one generation within the other (Dixon, 1998). This trait allows a quick response to environmental factors (e.g. the production of winged forms due to crowding and host plant nutrition), as their effects are simultaneously experienced by up to three generations (Dixon, 1998). Each female can produce some 30–60 offspring per generation dependant upon species and in the absence of a range of mortality factors (climatic, predation, disease), populations may undergo an exponential increase, resulting in literally billions of individuals (Harrington, 1994). We believe that the maintenance of a clonal lineage is fascinating in itself, where intraclonal genetic variation (genetic fidelity), along with ageing and longevity (genetic integrity) of the lineage, are essential factors in its continuity. Both are fundamental to a better understanding of evolution in asexual organisms. As genetic fidelity has been discussed at length by us elsewhere (Lushai and Loxdale, 2002; Loxdale and Lushai, 2003), in this paper, we primarily focus on the nature of the molecular mechanisms related to the genetic longevity of eukaryotic lineages, more especially with respect to changes in telomere regions of the chromosome. We then compare this phenomenon as described mainly for cells with that of asexual lineages of whole eukaryotic organisms. For the sake of clarity, the ideas expounded here are based on apomicts (not isolines) and, using suitable experimental regimes, may allow us to test alternatives to evolutionary theory pertaining to the persistence of lineages per se.

2. Mechanisms of genetic change 2.1. What of genetic fidelity? One of the prominent aspects of clonal lineage persistence is genetic fidelity, i.e. intraclonal variation. In our recent papers discussing this topic, our overall conclusion was that due to a plethora of dynamic, often rapid molecular mechanisms, the clone sensu stricto is not a perfect or ‘ideal’ form in the Aristotelian sense, just an image of such an ideal (Loxdale and Lushai, 2003). Hence, the semantic description suggesting that clones are ‘genetically identical’ is a biological improbability (Lushai et al., 2000; Lushai and Loxdale, 2002). In addition, some of the variance observed in clonal lineages undoubtedly has adaptive significance (Lushai et al., 2003). Therefore, in the present article, we only briefly discuss this aspect of lineage maintenance. Certainly in the light of the numerous ongoing changes within an asexual lineage, germline cells act as a partial barrier (‘bottleneck effect’), which overcome considerable individual somatic variation from being passed on to subsequent generations (e.g. Hughes, 1989). However, even with the presence of these safe-guarding cells, genome-wide mu-

tational changes tend to accumulate spontaneously in the lineage, and increase and fix with time (see also Schön and Martens, 2003). These can therefore increase the genetic load in subsequent generations, especially in small populations (i.e. Muller’s ratchet; Muller, 1964; Charlesworth and Charlesworth, 1997). Individuals are eliminated from the population if the deleterious mutation rate is higher than one per genome per generation, and such lineages would become extinct within a few generations of their appearance, e.g. the ‘Mutation Deterministic Model’ of Kondrashov (1988, 1998). The elimination of severely loaded individuals in a population may help to lower the genetic cost to that population with concomitant size reduction (Gabriel et al., 1993). Severe crashes in population have been described by the ‘mutational meltdown’ model when a lineage becomes selectively disadvantageous with time with consequent poor fitness and adaptability due to accumulated genetic load (Lynch et al., 1993; Lynch and Blanchard, 1998). (N.B. The discovery of a new class of DNA polymerases and their function broadens the perspective of stochastic genetic change in genomes to one that suggests the maintenance of some genetic change that, in particular environmental conditions, allows for rapid fitness increases; see Radman, 1999; Friedberg et al., 2002.) In empirical observations of asexual lineages of water fleas, Daphnia pulex (Leydig) (Cladocera), phenomena such as genomic mutational meltdown have been suggested as being responsible for the extinction of a clonal lineage in c. 100 generations, i.e. within a few years (Lynch et al., 1993). However, along with such mutation-based approaches, various selection models describe how asexual lineages are influenced by frequency-dependent relationships with heterogeneous environments (e.g. the ‘Tangled Bank’ hypothesis) or with natural enemies, the Red Queen hypothesis (e.g. Charlesworth, 1987; but see Jokela et al., 1997). Irrespective of the model adopted, lineage extinction cannot be ignored and is probably the general outcome for many clonal lines. The crux of the present article is lineage longevity or maintenance and although predictions of the durability of various lineages exist, albeit with considerable variation for animals, plants and fungi, i.e. 101 to 108 years (Lynch and Blanchard, 1998), a unifying molecular genetic mechanism of how lineages—both asexual and sexual—are in fact maintained is lacking. Hypothetically, sex has been considered essential to overcome genetic burdens accumulated with time and also, as a source of continued variability which allows adaptation to ever changing environments (see West et al., 1999 for a review). Even so, the maintenance of sex in spite of various factors such as the ‘two-fold cost of sex’ (Maynard-Smith, 1978; see also Crow, 1999) versus other lifecycle strategies like asexuality, remains an enigma (but see Avise, 1993). It could be that strategies like sex have a further role additional to overcoming genome wide mutational loads and producing genetic variability in the face of new environmental pressures. We suggest that this role has been tackled in

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

different ways by different biological systems, and although sex may excel in a long-term evolutionary sense for most organisms (Simon et al., 2003), it is not the only solution. The most remarkable of the alternative systems include ancient asexual lineages, more especially bdelloid rotifers and some darwinulid ostracods, where chromosomal recombination is largely absent from whole animal groups (Normark et al., 2003). These roles are quintessentially the underlying phenomena sustaining lineages through time. An example that highlights the ability of long-lived asexual lineages to persist has been given in a seminal study of asexual bacterial endosymbionts of insects. Lineages of these ancient prokaryote asexual organisms show the fast accumulation of mutations as expected in a small population, and seem to conform to Muller’s predictions (Moran, 1996). Yet these lineages have not suffered the expected extinction over the last 100+ million years, a result that the author suggests may indicate strong compensatory processes overriding the deleterious effects, or alternatively, may be due to selective sweeps that are too weak or difficult to detect. We herein postulate that the crucial genetic constraint that brings about the death of a genotype or lineage may not be related to a factor that necessarily operates across the genome, such as genetic load (here, we preclude observations of stochastic events amplified by enhanced energy sources like UV-radiation during artificially-induced exposures of experimental lineages). Rather, the observed longevity of these clonal lineages may result from genomic integrity maintained at a more specific location within the genome and/or, one associated with the function of that region (see also Redfield, 1999). We examine this point specifically for eukaryotic clonal lineages, and move away from the examination of only genome-wide stochastic events, which may provide clues to the factors controlling lineage longevity. Instead, the effects on specific regions of the genome in relation to extinction events are considered. A good candidate-site upon which there has been much research in recent years and that governs the long-term persistence of eukaryote clonal lineages are the physical ends of the chromosome, the telomeres. 2.2. Lineage longevity based on telomere functioning Telomeric activity has to date been elucidated using a number of systems—mammalian cell lines, budding yeasts, ciliates, nematodes and insects (Drosophila). Certain mammalian cell lines are known to senesce after a set number of proliferations (cell divisions) as described above. In contrast, cancerous cells are effectively immortal in vitro, e.g. HeLa cells. Such immortality is apparently related to high telomerase activity. For some while, it was thought that telomere length and activity were the major factors involved in cell growth, proliferation and ultimately senescence. It is now known that there are numerous vital components involved in various complex processes that influence cell lineage longevity and certainly, the balance of these other

261

components with the telomere–telomerase interaction is crucial. The telomeres are the physical ends of linear eukaryotic chromosomes. They were initially observed by Hermann J. Muller in 1938 and Barbara McClintock in 1941 working on different organisms. Muller coined the term ‘telomere’, from the Greek for ‘end’ (telos) and ‘part’ (meros) (McClintock, 1941; Muller, 1962; Greider and Blackburn, 1996). These specialised ribonucleoprotein complexes are composed of an RNA subunit involved in the protection, replication and stabilisation of the chromosome ends (McEachern et al., 2000; Blackburn, 2004). They comprise tandemly repeated DNA sequences which are highly conserved and of similar sequence in all organisms so far studied (TTAGGG in vertebrates; TTAGG in insects, but see Frydrychova and Marec, 2002), and are under control by a reverse transcriptase, telomerase, which is responsible for telomeric repeat extension (McEachern et al., 2000; Aisner et al., 2002; Hahn, 2002). In human primary cells, the catalytic core molecular component of the enzyme is known as human telomerase reverse transcriptase (hTERT) (Chan and Blackburn, 2002). Other proteins also seem to be involved in telomeric function, e.g. MRT-2 in the nematode Caenorhabditis elegans (Maupas) which acts as a DNA damage ‘checkpoint’ protein (Ahmed and Hodgkin, 2000; Blackburn, 2000; Hahn, 2002). As telomerase activity decreases, telomeres shorten by a process that seems to occur in normal senescing cells (Hayflick, 1997). Most normal human cells do not express the hTERT gene, due to one or more repressors (Ducrest et al., 2002). Only cancer cells have unusually high expression of hTERT and thereby telomerase activity, and their lineages are considered to be ‘immortal’ (e.g. Kim et al., 1994; Hayflick, 1997; Ducrest et al., 2002). Immortal lineages, whether cancerous or not, maintain telomeres of stable length as a result of this high telomerase titre (Aisner et al., 2002). Therein lies our analogy with the maintenance of ancient asexual lineages. In mammalian and yeast cells, telomere functioning as presently understood is a complex interaction of several components that are only just being elucidated (Blackburn, 2004). Thus for example, the relationship of telomere length (shortness) related to senescence in mammalian cell lines appears to be dependant on cell type. In addition, with regard to telomerase activity, both old and young cell lineages with different telomere lengths can still persist for some time together (Blackburn, 2000). Even so, functionality dependent on titre of telomerase and telomere length nevertheless appears to have some role as to whether cells proliferate or begin to slow down metabolically towards senescence. A model of cell senescence describes the stochastic switching between capped and uncapped chromosomes occurring in populations of cells of differing age (Blackburn, 2000, see their Figs. 1 and 2). In general, capping is necessary for preserving the physical integrity of the chromosome end, whilst regulated uncapping is a property of dividing

262

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

cells. During early cell division, most cells have relatively long telomeres, are capped and undergo fast proliferation (active cell cycling). As they age towards senescence, the model depicts that the ratio switches over and they then tend to have shorter telomeres which become, in terms of population frequency, progressively more uncapped. Uncapped telomeres can channel the DNA damage response able to repair them, and stimulate telomerase activity, which in turn stimulates cell division. However, if uncorrected for long, the uncapped state also triggers cell cycle arrest, i.e. such uncapped cells enter a state known as ‘replicative senescence’, whereupon most cells are arrested in terms of cell cycling so that proliferation slows and eventually ceases with senescence and cell death (see Blackburn, 2000 for a detailed overview). Even so, new data and the above model indicate that telomere length alone cannot be taken as an exact measure of how old a given cell lineage is. Rather, as discussed by Blackburn (2000), longer telomeres with more telomeric repeats and thus more protein binding sites, are more likely to switch towards capped structures than chromosomes with shorter telomeres. It seems that whilst telomere length per se does not always signify whether a telomere is functional, uncapping to produce a chromosome end that looks like a DNA break is necessary to trigger the response of telomerase and associated proteins to repair it. All this is intricately involved in cell regulation, which links telomeres and DNA damage signalling in cells, and in turn brings about important repair enzymes to act on the chromosome broken ends. Accordingly, a major effect in damage signalling is to ‘divert the DNA damage response into telomerase action rather than into DNA end-to-end joining’ (Blackburn, 2000). The activation of hTERT is the limiting step for induction of telomerase in most cell lineages studied to date. According to Hahn (2002), cells that express hTERT allow telomeres to respond in the same manner as pre-senescent, non-immortalised cells even in the face of serum deprivation and gamma-radiation. Consequently, such cells are able to bypass replicative senescence. Studies have also shown that the telomeric regions of chromosomes are often heterochromatic and thereby posses a high degree of DNA folding. In humans, protection of chromosome ends primarily depends on the telomeric protein TRF2 and formation of a high order structure, the telomeric loop or t-loop (De Lange, 2002). It seems from some experiments that structural folding of the terminal regions of the telomere can contribute to telomere capping (Chan and Blackburn, 2002). Loss of telomere capping can lead to chromosome non-disjunctions as well as the potential for loss of genetic information through degradation of the terminal region of the chromosome (Blackburn, 2000; Cooper, 2000; De Lange, 2002). Similarly, in cell lineages, mutations in the DNA damage repair proteins required for telomere maintenance (structural modulation and/or to form a suitable substrate for the enzyme), including deletion of the gene/s themselves, cause shortening of the telomeres and replicative

senescence (Chan and Blackburn, 2002). The importance of the integrity of these sites therefore seems to be fundamental to their function, and is the main reason why we here stress that stochastic mutational events that fix in germlines in such regions may be more important than genome-wide changes from the perspective of lineage load. Utilising the Blackburn model as an indicator of ageing, we suggest that a suitable molecular assay could identify, on the basis of a proportional index of capped to uncapped telomeres, the stage of senescence in an individual or test lineage, i.e. that from the population of cells analysed, obtained from a whole homogenate of the study organism, a ‘norm’ of capped/uncapped telomere state may be produced (see Section 3). In Drosophila and other Diptera (true flies), other mechanisms are involved in telomere elongation, i.e. long retrotransposons, which transpose to the ends of the chromosomes (Biessmann et al., 1997). It is possible that the large complex-sequence repeats of the telomeric regions may form a nucleosomally based, local heterochromatin structure that stabilises the telomeres (Chan and Blackburn, 2002) and, as such, chromosome capping involving specific telomeric sequences is not essential in these insects. Such observations suggest that any form of control in these regions will not necessarily be uniform and that compensatory mechanisms may well be species specific (see also Frydrychova and Marec, 2002). There are other confounding factors that will blur the correlation of telomere activity on longevity and these are briefly described here. Unfortunately, some of these may not be so readily tested in the experimental procedures described later for aphids. In eukaryotes, post mitotic cells will initially attempt to replace dead cells; however, this can eventually be impaired as a result of exhaustion of cell renewal related to replication associated, or stochastic damage associated, telomere shortening. One of the factors affecting such damage is as a consequence of small reactive molecules, i.e. reactive oxygen intermediates (ROIs), as detailed by Burkle (2001). These occur during normal cellular metabolism and are related to vital cellular functions. Damage to macromolecules can occur to proteins and lipids, although more importantly, to mitochondrial and nuclear DNA. Cellular dysfunction is manifest in a variety of ways, including genetic instability, cell differentiation and death. Experimental evidence shows that lowering production of ROIs or improving cellular antioxidative defences slows down the ageing process (Burkle, 2001). Besides cell-specific ageing process observed in vitro as above, ageing is well documented in the whole organism in vivo. It has a plethora of causes ranging from known genes in yeast, nematodes, mice and other well studied eukaryotes and is often revealed due to a mutation in a particular candidate gene related to some important biochemical/physiological process (see Curtsinger et al., 1995). Such processes include apoptosis, which is programmed cell death occurring during normal development and regulation

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

of the immune response, e.g. the daf-2 gene in C. elegans, which causes adults to live twice as long as the wild type. Some such genes are certainly involved in the regulation of various biological parameters or are themselves regulatory. Otherwise, they may be involved in inhibiting apoptosis via several different pathways: antioxidants; immunological or heat-shock proteins produced in response to heat and other environmental stresses. There appears to be a link between ageing and heat-shock proteins and certainly in C. elegans, mutants that have increased lifespan have increased thermotolerance (Lithgow et al., 1995). In other instances, ageing responses are more plastic, as noted in Drosophila, where lifespan is described as a typical quantitative character that varies on a continuous scale and responds to selection, lineage and various environmental effects. However, whilst longevity is influenced by both physical traits such as temperature, humidity, culture conditions and by physiological factors like reproductive status, mating and passage of fluids by flies during mating, longevity is seen to have low heritability, as with other life history traits (Curtsinger et al., 1995). Although the traits from these latter ageing studies may be difficult to test within aphid lineages, there is certainly opportunity for prospective researchers to verify telomere function in the lineages which describe changes in longevity. Below, we describe experiments with illustrations of expected results based on our hypothesis that telomere function plays a central role in the longevity of lineages, as well as that of individuals.

3. Testing for telomere function in lineages For the proposed experiments, we considered two independent observations: (1) research on the asexual phase of the ciliate, Tetrahymena thermophila Nanney and McCoy showing that a functional telomere is required for normal separation of sister chromatids during mitotic anaphase, and that mutant telomeres cause chromosome ends to become inseparable, ending the lineage (Kirk et al., 1997); (2) in the uncapped state, certainly as shown in yeasts, homologous recombination (Shibata, 2001) resets the telomere length and associated capped state, even without the requirement of telomerase (Lundblad, 2002). We unite these two observations with the situation found in holocyclic aphid lineages where sex occurs annually after some 14 asexual generations, triggered by environmentally-induced factors. By extrapolating these trends, we suggest that the changing state of telomeres as found in ageing cell lineages is possibly analogous and central to the maintenance of asexual lineages and that the role of recombination may include the ability of ‘resetting’ telomere function, a premise we feel is testable using holocyclic aphid lineages. The telomeric repeat TTAGG is known to occur in the obligate asexual aphid, for example, the snapdragon aphid, Myzus antirrhinii (Macchiati) (Spence et al., 1998). It is

263

therefore plausible to study the importance of telomere form and function in this interesting group of insects that usually has both asexual and sexual lifecycles within a given species (Blackman and Eastop, 2000). This will allow determination of the relevance of telomere function on lineage longevity in the same species, over and above the functional replication of isoline studies as in Drosophila and nematodes, i.e. C. elegans. The proviso is that molecular assays performed on whole organism preparations will allow capped/uncapped indices of telomeres to be traced in given individuals at pre-specified times, i.e. 1–2 days after becoming adults, and from across generations within selected lineages (for details of molecular methodology, see Jacob et al., 2001 and Baerlocher et al., 2002). As described in Section 1, aphids are wonderfully complex organisms, having multiple lifecycles with multiple phenotypes. They can exhibit up to seven distinct phenotypes, either within the main holocyclic lifecycle or variants thereof (Fig. 1; see also Moran, 1994 and Simon et al., 2002 for further details). The forms often have very different physiology and ecology in relation to one another, even though they are parthenogenetically linked in a lineage. In such clonal lineages, for a given generation, numerous individuals are born within a day. For our purposes, forms 3 and 4 (Fig. 1) are most appropriate to set up a facultative asexual lineage. Therefore, individuals from one generation can be isolated to set up parallel sub-lineages that can be tested under different treatments, in the same time-line. Alternatively, if a test for individual senescence in a generation is required, the individuals in a generation born at approximately the same time can be isolated and reared, and later sequentially sacrificed through time to study telomere changes as ageing occurs. Individual female aphids, e.g. the grain aphid, Sitobion avenae (F.), mature in approximately 12 days and will continue to reproduce through a process of viviparous parthenogenetic reproduction for a further 10–15 days after which they will remain alive for another 5–10 days on average. In the following scheme, we describe trends in capped telomere frequency (i.e. cell population norms), that we would expect for different experimental aphid lineages as a result of their lifecycle character, i.e. hypothetical1 ancient asexuals, sexuals, facultative and obligate asexuals. We hypothesise that based on a working molecular assay (from the DNA extraction of whole individuals), capped telomere frequency will: Be maintained over generations (1 to say 200) for ancient asexuals (Fig. 2 (i)) and for sexuals (Fig. 2 (ii)). It is thought that even the slope of these will diminish, but spanning millions of years this will be indistinguishable over experimental time frames. 1 Hypothetical, as true ancient aphid asexuals still need to be verified, but the capped telomere frequency from suspected ancient asexuals, such as bdelloids, could be used.

264

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

Fig. 1. Schematic representation of facultative (holocyclic) and obligate (anholocyclic) asexual life cycles in a hypothetical aphid species (figure adapted from Field and Blackman, 2003). A sexual egg hatches to produce the stem mother (1) of the facultative asexual lineage. After the spring migrant (2), this can then be maintained as apomictic parthenogens in summer forms (3 and 4) provided the right environmental conditions are met: e.g. light/dark 16 h:8 h, 15 ◦ C. In general, obligate asexual lifecycles are ‘locked’ into forms 3 and 4 irrespective of environmental conditions (although rare, androcyclic or intermediate lifecycles may occur in a species, see Section 1). In facultative asexuals, summer forms can be induced to produce the autumn migrant (5) and male (6) in appropriate conditions, e.g. light/dark 8 h:16 h, 15 ◦ C. These conditions will also induce the autumn migrant to give birth to the sexual female (7). Sexuals mate forming a sexual egg.

Frequency of capped telomeres

Diminish over generations (1–200) in both facultative and obligate asexuals (i.e. not from an ancient asexual lineage) (Fig. 2 (iii)). In Fig. 2, the actual level of slopes of ancient asexuals versus sexuals is arbitrarily represented and separated for visual clarity. Suffice to say that a high frequency of capped telomeres would be expected over generations, establishing lineage longevity. In contrast, some form of observable de-

cay pattern may be expected in both of the facultative and obligate asexual lineages over generations to the point where they die out. Ideally, these two lineages will have no difference in trends as asexual reproduction in both is apomictic. A newly established facultative asexual lineage (see Fig. 1) is expected to have a higher initial frequency than that of an obligate asexual lineage, where the history of the lineage would be unknown. Even so, for both lineage types,

Ancient asexual (i) Sexual (ii)

Best fit slopes

Facultative/obligate asexual(iii)

1

100

200

Generations Fig. 2. Hypothetical best fit slopes showing frequency of capped telomere cell population norms, measured using a molecular essay on three classes of lineage: (i) ancient asexuals; (ii) sexuals; and (iii) facultative/obligate asexuals. Measurements describe essays based on individuals at preset ages (e.g. 1–2 days after turning adult) over generations (1–200), or as necessary. This schematic only aims to show relative abundance of capped telomeres between the lineages, and does not suggest that ancient asexuals will necessarily have a greater capped telomere frequency than sexual lineages, for example.

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

the extinction of lineages should correlate strongly with the decline in the frequency of capped telomeres. If reported trends are similar for both such lineages, then later experiments could utilise the facultative asexual lineage only for the sake of simplicity. The null hypothesis is represented schematically in Fig. 3a. Here individuals are tracked again in terms of capped telomere frequency, but only through individual lifespans using both sexuals (Fig. 3a (i)) and facultative asexuals (Fig. 3a (ii)). This will test the assumption that capped telomere frequency indeed diminishes as an individual senesces and eventually dies. If this diminution does not occur, then the hypothesis will of course be found wanting. Again, the slopes and positioning described between sexual

and facultative asexual lineages are arbitrary, but in theory at least, both should follow a similar trend. An organism like hydra could add another dimension to this individual longevity test acting as a negative control to ‘complete’ senescence leading to death. The wave pattern schematically describes suspected cycles through ‘degeneration’ and ‘regeneration’ of telomere states for such an organism, which in itself is a fascinating study. Fig. 3b describes a further important scenario that can be tested empirically using facultative asexual aphid lineages. Sexual forms can be readily induced in such parthenogenetic female lineages by the manipulation of night length and ambient temperature conditions (e.g. light/dark, 8 h:16 h, 15 ◦ C, Fig. 1). Here, it is hypothesised that in an ageing facultative

Frequency of capped telomeres

Best fit slopes Hydra-type organism

Sexual (i)

apoptosis

1 (a)

2

Facultative/obligate asexual (ii)

3

Frequency of capped telomeres

Individual generations

Facultative asexual through recombination (ii, see Fig. 1 shaded area)

Point of mating

X

Best fit slopes Facultative asexual control (i)

1 (b)

265

100

200

Generations

Fig. 3. (a) This describes the best fit slopes of capped frequency changes that would be expected in: (i) sexual and (ii) facultative/obligate asexual lineage individuals over a generation and will confirm that the molecular essay is sensitive to telomere changes over time, i.e. leading to senescence and finally apoptosis. As clonal organisms (aphid lines) will be used to produce replicate samples that can be sacrificed over time, the curve will represent the collation of several insect telomere frequency measures representing the same generation in time. A negative control to apoptosis may be represented by the telomere frequency count in organisms with fission replication, like hydra. (b) A schematic of the expected curves for telomere frequency in: (i) a maturing facultative asexual lineage ‘control’ and (ii) a replicate facultative asexual lineage induced to produce sexuals and mated resulting in a new lineage after sexual egg hatch (see Fig. 1 hatched area). This test will indicate if recombination can reset telomere frequency to levels before the original asexual decline.

266

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

asexual lineage, with concomitant decline in the frequency of capped telomeres (Fig. 3b(i)), a new lineage will be produced following successful mating and subsequent sexual egg hatch, with a restored frequency of capped telomeres, represented by Fig. 3, ‘x’ and lineage Fig. 3b(ii). The following tests can also be carried out using aphids. Replication in an apomictic clonal lineage is easily carried out by isolating several sub-lineages from individuals born at a similar time (during a day) in a given generation. Each sub-lineage can be placed under different experimental regimes and studied over generations. Thus, the effects of topically applied or artificially elevated levels of hTERT-like proteins found in invertebrates (e.g. MRT-2), chemical agents such as porphyrins and AZT (Izbicka et al., 1999), and ROIs, etc., which inhibit telomerase function, can be followed in synchronous and genetically similar lineages, a single individual at a time and over several generations. In the event that an experimental asexual lineage (facultative or obligate) starts to die out over time, the frequency of capped telomeres and/or the functional telomere region may be studied by ‘molecular autopsy’ for symptoms and possible causation. The well-documented range of mutations operating in the ‘checkpoint kinase pathways’ that respond to DNA damage or replication problems causing modest alterations in telomere length and TPE (telomere position effect—found in yeasts where the telomeric-complex act as a repressive chromatin domain on the transcription of nearby genes; Cooper, 2000) could be tested for. Also, some checkpoint-defective cells exhibit ‘a striking loss of telomeric DNA with ensuing senescence identical to a telomerase dysfunctional phenotype’ (Cooper, 2000) and this could again be followed up.

4. Discussion Since mammalian cell lineages proliferate for about 60 generations before senescence and death, a facultative asexual aphid in the field only has to undergo a quarter as many mitotic divisions before recombination in the autumn, that is to say around 14 asexual generations. In the case of an obligate asexual aphid, about 3 years of constant asexual propagation would be expected before demise of the lineage, which is a trend noted within asexual Daphnia populations before mutational meltdown (Lynch et al., 1993). The occurrence of sex once a year for most facultative asexual aphid species is one of the pivotal reasons for the consideration that there is a connection between the persistence of asexual lineages and cycles of recombination within them. The retention of the sexual phase involving the laying of overwintering eggs has been described as not just an overwintering survival ploy (e.g. Dixon, 1998), but also as an evolutionary function, to purge the genome of deleterious accumulated mutations (Wilson et al., 1999; Normark and Moran, 2000), as well as to maintain genetic diversity (Lushai et al., 2003). We now add that perhaps another crucial role of sex, even

rare sex, is that it contributes to the maintenance of lineages per se, its effect acting via telomere function. The aim of the aforementioned investigations is to be able to test this hypothesised telomere operation. Therefore, at the simplest level, we propose adoption of an aphid model to confirm some of the trends already noted in other organisms, for instance, that a molecular essay will show that telomere function-related apoptosis leads to death of the individual organism. The eukaryotic sexual individual, which begins life as a single fertilised cell, and which then grows into a multicellular organism (itself producing gametes), ultimately senesces and dies, just as an asexual individual does. A successful molecular essay will therefore be able to record trends as noted in Fig. 3a for both lifecycle types. This approach should verify that the soma of sexual and asexual lineages behaves in exactly the same manner once the biological clock starts ‘ticking’ from the point of cell differentiation following fusion of the specialised germ cells to produce the zygote. The next point revealed would be to observe whether the germ cells within a lineage/s are in effect ‘switched off’, or ‘frozen’ from the general somatic senescence of the ageing individual. This must be the case to some extent for all lifecycle types, as it may be surmised that successful offspring have functional telomeres, even though the preceding generation has duly aged and died. It has been suggested by some that our experimental methodology will only confirm that the germline cells are the source of lineage longevity. Indeed, we know that recombination does occur between repeated sequences during mitosis and in the germline of parthenogenetic organisms and as telomeres are highly repetitive, it is likely that some such homologous recombination occurs in these regions as well. However, how efficient facultative/obligate asexual lineages are in this respect may hopefully also be elucidated by the experiments proposed. As Fig. 2 shows, we assume that ancient asexual and sexual lineages will have compensatory mechanisms allowing the lineages to maintain a high level of capped telomere frequency over time, whereas facultative/obligate asexual lineages will show a more significant tapering off in this frequency with time. In other words, these latter lineages will eventually die out in a way similar to that described for mutational models or sex-linked theory, but for a very different reason. It is true that even if these trends are shown, understanding of just how telomeres avoid degradation through the different germlines is further required, but at least the experiments will allow the importance of the telomere site to be correlated with lineage longevity, even if not to directly establish it. As discussed, our view is that telomeres slowly degrade in the germline of facultative/obligate asexuals due to the omission of an advantage allowed by, for example, meiotic recombination, and we feel that the ancient asexuals and sexuals are efficient, although possibly in very different ways, in performing this function as result of a more robust mechanism. Therefore, the latter are able to undergo processes

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

which reset the telomere length and capping from generation to generation more efficiently. Empirical findings based on the experiments detailed in Fig. 3b may ascertain this. If telomere frequency is indeed ‘reset’ (represented by Fig. 3b (x)) in a mature line of facultative asexuals after passing through a recombination step, the inference is of course that this is a primary function of recombination. This being so, then such experimentation may allow elucidation and understanding of more than just a mere ‘black box’ depiction of the germline/telomere function on lineage persistence. The application of this approach to aphid taxa where obligate asexuals are thought to be prevalent versus other taxa which have predominantly facultative asexual lifecycles (Blackman et al., 2000) may also allow different telomere control mechanisms to be elucidated. This would be particularly pertinent as some clonal lineages which appear to persist in the total absence of recombination, e.g. some asexual ciliate strains, Tetrahymena, and the shallot aphid mentioned earlier, suggest that recombination cannot be entirely necessary for this function, unless that is, sex does very occasionally occur. Rare sex is certainly alluded to in predominately obligate asexual aphid species (Normark, 1999). The absence of males may be as much a population sampling effect as a reality. Occasional recombinational events may be all that is required in asexual populations to allow descendants to thrive in competition with other asexual lineages that have not as yet recombined. Perhaps we can draw a simple analogy to the stochastic processes for the switch in ageing cell lineages where capped telomeres are replaced by uncapped ones leading to their death. This could be equated with recently recombined versus persisting asexual lineages, respectively, with the suggestion that stochastic processes along with natural selection marshal clonal lineages which vie for dominance within a population. If some obligate asexual aphid populations persist for long periods, as indicated in recent studies of S. avenae (Delmotte et al., 2001), a population consisting of billions of females may well produce males very occasionally (=androcycly, see Section 1), which in turn mate with females from sexual lines of the same species (see Delmotte et al., 2001). If so, such clonal lineages are perhaps only ‘images of immortality’ and in reality, are occasionally rejuvenated lineages that as a result of stochastic processes involving recombination, propagation, decline and replacement, or selective sweeps of particularly ‘fit’ genotypes, appear to continue through time. Alternatively, and even more rarely, they may have developed, like ancient asexuals, compensatory mechanisms that maintain telomere function and allow truly immortal asexual lineages to persist through the aeons. Such mechanisms may include preservation of the telomeres from the influence of control enzymes or mutators. Very interestingly in this respect, bdelloid rotifers appear not to have reverse transcriptases (telomerase itself is such an enzyme) and also reveal a dearth of LINE-like and gypsy-like

267

retrotransposons throughout their genomes (Arkhipova and Meselson, 2000). In groups of ancient asexuals like the darwinulid ostracods, the species Darwinula stevensoni Brady and Robertson shows little or no genetic variation at certain sites of high molecular variation (rDNA), indicative of mechanisms of slow molecular evolution with a propensity to highly efficient DNA repair (Schön and Martens, 1998). In this group, this particular attribute may be causal to continued telomere functioning. Research along the lines described above for aphids would certainly promote a better understanding of telomere function in these remarkable lineages, i.e. capped/uncapped states, chromosome structure, folding, etc., along with hTERT-type interaction with telomerase. Examination of telomere functioning in ancient asexuals and organisms displaying different lifecycle types may detail the variety of group-specific mechanisms that control telomere function other than the more ubiquitous method based on recombination.

5. Conclusions If the molecular markers chosen allow differentiation of telomere length in aphids, by allowing some capped frequency index to be generated, then the hypothesis that telomere function is associated with lineage persistence over generations can indeed be tested empirically. If the hypothesis describes or denies the predictive schematics (Figs. 2 and 3) of capped/uncapped frequency in lineages over time, then the theory can be qualified. What of concepts of immortality? In short, there are two components to clonality or clonal immortality. Firstly, the genetic clone. In all fairness, this probably does not exist in terms of strict genetic fidelity, although this does not mean that a ‘clonotype’ descended from a stem mother (e.g. in aphids, the first asexual female aphid after egg hatch) may not persist for a very long time. Secondly, as individuals within an asexual lineage senesce, there must be mechanisms that prevent a similar process between generations, at least in the short term, maintaining lineage longevity. Investigating these processes along the lines suggested with aphids with multiple complex lifecycles and other apomictic eukaryotic lineages such as Daphnia, will provide deeper insights into these mechanisms and allow the elucidation of the physiological factors influencing this aspect of lineage immortality. Confirmation of the role that recombination in sexuals or compensatory mechanisms in ancient asexuals play in resetting telomere functioning will be illuminating. It may also allow re-evaluation of the function of sex, perhaps over and above the emphasis of its use in ridding the genome of mutational loads and maintaining high levels of adaptability in constantly changing environments (Lushai et al., 2003). The presence of other lifecycle strategies that persist in nature even though sex is ubiquitous is in itself indicative of the failings of the present assumptions in evolutionary genetics.

268

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269

Acknowledgements We thank Drs. Teresa Crease and Isa Schön for their critical appraisal of earlier versions of the manuscript and Professor Harriet Smith-Somerville for providing information on Tetrahymena. H.D.L. was supported by the Department of Environment, Food and Rural Affairs (DEFRA), UK. Rothamsted Research receives grant-aided support from the BBSRC.

References Ahmed, S., Hodgkin, J., 2000. MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159–164. Aisner, D.L., Wright, W.E., Shay, J.W., 2002. Telomerase regulation: not just flipping the switch. Curr. Opin. Genet. Dev. 12, 80–85. Arkhipova, I., Meselson, M., 2000. Transposable elements in sexual and ancient asexual taxa. Proc. Natl. Acad. Sci. U.S.A. 97, 14473–14477. Avise, J.C., 1993. The evolutionary biology of ageing, sexual reproduction and DNA repair. Evolution 47, 1293–1301. Baerlocher, G.M., Mak, J., Tien, T., Lansdorp, P.M., 2002. Telomere length measurement by fluorescence in situ hybridisation and flow cytometry: tips and pitfalls. Cytometry 47, 89–99. Biessmann, H., Walter, M.F., Mason, J.M., 1997. Drosophila telomere elongation. Ciba Foundation Symp. 211, 53–67. Blackburn, E.H., 2000. Telomere states and cell fates. Nature 408, 53–56. Blackburn, E.H., 2004. Telomeres and telomerase. Philos. Trans. R. Soc., vol 359, in press. Blackman, R.L., 1979. Stability and variation in aphid clonal lineages. Biol. J. Linnean Soc. 11, 259–277. Blackman, R.L., Eastop, V.F., 2000. Aphids on the World’s Crops: An Identification and Information Guide, second ed. Wiley, Chichester, UK. Blackman, R.L., Spence, J.M., Normark, B.B., 2000. High diversity of structural heterozygous karyotypes and rDNA arrays in parthenogenetic aphids of the genus Trama (Aphididae: Lachninae). Heredity 84, 254– 260. Burkle, A., 2001. Mechanisms of ageing. Eye 15, 371–375. Chan, S.W., Blackburn, E.H., 2002. New ways not to make ends meet: telomerase, DNA damage proteins and heterochromatin. Oncogene 21, 553–563. Charlesworth, B., 1987. Red queen versus tangled bank models. Nature 330, 116–117. Charlesworth, B., Charlesworth, D., 1997. Rapid fixation of deleterious alleles can be caused by Muller’s ratchet. Genet. Res. 70, 63–73. Cooper, J.P., 2000. Telomere transitions in yeast: the end of the chromosome as we know it. Curr. Opin. Genet. Dev. 10, 169–177. Crow, J.F., 1999. The omnipresent process of sex. J. Evol. Biol. 12, 1023–1025. Curtsinger, J.W., Fukui, H.H., Khazaeli, A.A., Kirsher, A., Pletcher, S.D., Promislow, D.E.L., Tatar, M., 1995. Genetic variation and aging. Ann. Rev. Genet. 29, 553–575. De Lange, T., 2002. Protection of mammalian telomeres. Oncogene 21, 532–540. Delmotte, F., Leterme, N., Bonhomme, J., Rispe, C., Simon, J.-C., 2001. Multiple routes to asexuality in an aphid. Proc. R. Soc. Lond. B Biol. Sci. 268, 2291–2299. Dixon, A.F.G., 1998. Aphid Ecology, second ed. Chapman and Hall, London. Ducrest, A.-L., Szutorisz, H., Lingner, J., Nabholz, M., 2002. Regulation of the human telomerase reverse transcriptase gene. Oncogene 21, 541–552.

Field, L.M., Blackman, R.L., 2003. Insecticide resistance in the aphid Myzus persicae (Sulzer): chromosome location and epigenetic effects on esterase gene expression in clonal lineages. Biol. J. Linnean Soc. 79, 107–113. Friedberg, E.C., Wagner, R., Radman, M., 2002. Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science 296, 1627–1630. Frydrychova, R., Marec, F., 2002. Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera). Genetica 115, 179–187. Gabriel, W., Lynch, M., Burger, R., 1993. Muller’s ratchet and mutational meltdown. Evolution 12, 627–640. Greider, C.W., Blackburn, E.H., 1996. Telomeres, telomerase and cancer. Sci. Am. 274 (February), 80–85. Hahn, W.C., 2002. Immortalization and transformation of human cells (mini-review). Mol. Cells 13, 351–361. Harrington, R., 1994. Aphid layer (letter). Antenna (Bull. R. Entomol. Soc.) 18, 50. Hayflick, L., 1965. The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res. 37, 614–636. Hayflick, L., 1997. Mortality and immortality at the cellular level. A review. Biochemistry (Mosc.) 62, 1180–1190. Hughes, R.N., 1989. A Functional Biology of Clonal Animals. Chapman and Hall, London, UK. Izbicka, E., Nishioka, D., Marcell, V., Raymond, E., Davidson, K.K., Lawrence, R.A., Wheelhouse, R.T., Hurley, L.H., Wu, R.S., VonHoff, D.D., 1999. Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos. Anticancer Drug Des. 14, 355–365. Jacob, N.K., Skopp, R., Price, C.M., 2001. G-overhang dynamics at Tetrahymena telomeres. EMBO J. 20, 4299–4308. Jokela, J., Lively, C., Fox, J.A., Dybdahl, M.F., 1997. Flat reaction norms and “frozen” phenotypic variation in clonal snails (Potamopyrgus antipodarum). Evolution 51, 1120–1129. Kim, N.W., Piatyszek, M.A., Prowe, K.R., Harley, C.B., West, M.D., 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015. Kirk, K.E., Harmon, B.P., Reichardt, I.K., Sedat, J.W., Blackburn, E.H., 1997. Block in anaphase chromosome separation caused by telomerase template mutation. Science 275, 1478–1481. Kondrashov, A.S., 1988. Deleterious mutations and the evolution of sexual reproduction. Nature 336, 435–441. Kondrashov, A.S., 1998. Measuring spontaneous deleterious mutation process. Genetica 103, 183–197. Lithgow, G.J., White, T.M., Melov, S., Johnson, T.E., 1995. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress. Proc. Natl. Acad. Sci. U.S.A. 92, 7540–7544. Loxdale, H.D., Lushai, G., 2003. Rapid changes in clonal lineages: the death of a ‘sacred cow’. Biol. J. Linnean Soc. 79, 3–16. Lundblad, V., 2002. Telomere maintenance without telomerase. Oncogene 21, 522–531. Lushai, G., Loxdale, H.D., 2002. The biological improbability of a clone (mini-review). Genet. Res. 79, 1–9. Lushai, G., Loxdale, H.D., Maclean, N., 2000. Genetic diversity of clonal lineages. J. Reprod. Dev. (Suppl.) 46, 21–22. Lushai, G., Loxdale, H.D., Allen, J.A., 2003. The dynamic clonal genome and its adaptive potential. Biol. J. Linnean Soc. 79, 193–208. Lynch, M., Blanchard, J.L., 1998. Deleterious mutation accumulation in organelle genomes. Genetica 103, 29–39. Lynch, M., Burger, R., Butcher, D., Gabriel, W., 1993. The mutational meltdown in asexual populations. J. Heredity 84, 339–344. Mandrioli, M., Manicardi, G.C., Bizzaro, D., Bianchi, U., 1999. NOR heteromorphism within a parthenogenetic lineage of the aphid, Megoura viciae. Chromosome Res. 7, 157–162. Maynard-Smith, J., 1978. The Evolution of Sex. Cambridge University Press, Cambridge. McClintock, B., 1941. The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234–282.

H.D. Loxdale, G. Lushai / Infection, Genetics and Evolution 3 (2003) 259–269 McEachern, M.J., Krauskopf, A., Blackburn, E.H., 2000. Telomeres and their control. Ann. Rev. Genet. 34, 331–358. Moran, N.A., 1994. Adaptation and constraint in the complex life cycles of animals. Ann. Rev. Ecol. Syst. 25, 573–600. Moran, N.A., 1996. Accelerated evolution and Muller’s ratchet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. U.S.A. 93, 2873–2878. Muller, H.J., 1962. The Remaking of Chromosomes. Studies of Genetics: The Selected Papers of H.J. Muller. Indiana University Press, Bloomington, USA, pp. 384–408. Muller, H.J., 1964. The relation of recombination to mutational advance. Mutat. Res. 1, 2–9. Normark, B.B., 1999. Evolution in a putatively ancient asexual aphid lineage: recombination and rapid karyotype change. Evolution 53, 1458–1469. Normark, B.B., Moran, N.A., 2000. Testing for the accumulation of deleterious mutations in asexual eukaryote genomes using molecular sequences. J. Nat. Hist. 34, 1719–1729. Normark, B.B., Judson, O., Moran, N., 2003. Genomic signatures of ancient asexual lineages. Biol. J. Linnean Soc. 79, 69–84. Powell, G., Hardie, J., 2000. Host-selection behaviour by genetically identical aphids with different plant preferences. Physiol. Entomol. 25, 54–62. Radman, M., 1999. Mutation-Enzymes of evolutionary change. Nature 401, 866. Rawes, V., Kipling, D., Kill, I.R., Faragher, R.G.A., 1997. The kinetics of senescence in retinal pigmented epithelial cells: a test for the telomerase hypothesis of ageing? Biochemistry (Mosc.) 62, 1291–1295.

269

Redfield, R.J., 1999. A truly pluralistic view of sex and recombination. J. Evol. Biol. 12, 1043–1046. Shibata, T., 2001. Functions of homologous DNA recombination. RIKEN Rev. 41, 21–23 (http://www.riken.go.jp/lab-www/library/publication/ review/pdf/No 41/41 021.pdf). Schön, I., Martens, K., 1998. Opinion: DNA repair in an ancient asexual— a new solution for an old problem? J. Nat. Hist. 32, 943–948. Schön, I., Martens, K., 2003. No slave to sex. Proc. R. Soc. Lond. B Biol. Sci. 270, 827–833. Simon, J.-C., Rispe, C., Sunnucks, P., 2002. Ecology and evolution of sex in aphids. Trends Ecol. Evol. 17, 34–39. Simon, J.-C., Delmotte, F., Rispe, C., Crease, T., 2003. Phylogenetic relationships between parthenogens and their sexual relatives: the possible routes to parthenogenesis in animals. Biol. J. Linnean Soc. 79, 151–163. Spence, J.M., Blackman, R.L., Testa, J.M., Ready, P.D., 1998. A 169-base pair tandem repeat DNA marker for subtelomeric heterochromatin and chromosomal rearrangements in aphids of the Myzus persicae group. Chromosome Res. 6, 167–175. Tomiuk, J., Wöhrmann, K., 1982. Comments on the genetic stability of aphid clones. Experientia 38, 320–321. West, S.A., Lively, C.M., Read, A.F., 1999. A pluralist approach to sex and recombination. J. Evol. Biol. 12, 1003–1012. Wilson, A.C.C., Sunnucks, P., Hales, D.F., 1999. Microevolution, low clonal diversity and genetic affinities of parthenogenetic Sitobion aphids in New Zealand. Mol. Ecol. 8, 1655–1666.