A longstanding debate in evolutionary biology concerns how species of increasing structural complexity maintain their capacity for genetic variation—and, hence, adaptation and divergence—despite a predictably increasing need for genetic fidelity (Gulick 1893; Gould JL and Gould CG 1997). Relevant to this conflict, Cope's rule postulates that increasing body size creates a short-term reproductive advantage for the individual organism (Kingsolver and Pfennig 2004) while worsening long-term extinction risk for the clade (Van Valkenburgh et al. 2004; Hone and Benton 2005). This trade-off suggests that higher evolving organisms are subject to a progressive “Red Queen”–type clash between intensifying negative selection for phenotypic stability and weakening positive selection for genotypic variability (Markov 2000)—consistent with the modest proportion (0.03%) of coding sequence estimated to have been positively selected in humans, when compared with that negatively selected (2.5–5%) in humans or positively selected in simpler species such as Drosophila (20%) (Ponting and Lunter 2006). Indeed, prevailing theory teaches that most genetic novelty results from fixation of random (nonadaptive) drift affecting neutral (Kimura 1968) or near-neutral (Ohta 1998) alleles, rejecting the Lamarckian doctrine that environmental pressures can drive (i.e., not merely fix) beneficial mutations.

In previous work, we showed that silent mutations may nonrandomly affect intragenic sites of differing functional importance (Epstein et al. 2000; Lin et al. 2003) and that such mutational patterns vary with both strand-specific transcription-related DNA repair (Tang et al. 2006) and gene expression levels (Tang and Epstein 2007). It therefore remains plausible that ambient stressors such as heat (Maresca and Schwartz 2006), starvation (Hastings et al. 2004), inflammation (Blanco et al. 2007; Lavon et al. 2007), toxins (Salnikow and Zhitkovich 2008), free radical injury (Cerda and Weitzman 1997), or other sources of DNA damage (Ponder et al. 2005) could modify gene transcription and thus alter the rate of mutations affecting fitness (Galhardo et al. 2007)—including the occasional generation of beneficial mutations (Monk 1995; Elena and de Visser 2003; Nei 2005). Clues favoring this inducible (adaptive) evolutionary paradigm over neutrality for metazoan genomes—as is already accepted for bacterial (Ponder et al. 2005; Cirz and Romesberg 2007) and plant genomes (Galloway and Etterson 2007)—include faster-than-expected rates of phenotype acquisition, close temporal correlation with environmental changes, proof of improved fitness, or convergence (Levasseur et al. 2007).

A mechanism for such non-Darwinian genomic plasticity has been suggested in recent times by the discovery of heritable epigenetic changes capable of reprogramming developmental and adult gene expression (Martin et al. 2005; Morgan et al. 2005), coupled with the predisposition of such changes to cause germ line mutations (Cooper and Krawczak 1989) or postzygotic mosaicism (Ohlsson et al. 1999) that sometimes cause disease (Andrews et al. 1996; Smith and Hurst 1998; Esteller et al. 2001). The frequency of germ line epimutations or imprinting errors—estimated to be an order of magnitude higher than that of germ line mutations (Horsthemke 2006)—can be either environmentally regulated (Dolinoy and Jirtle 2008), as illustrated by the inducibility of spermatogonial stem cell DNA hypermethylation by air pollution (Yauk et al. 2008), or parentally age dependent (Oakes et al. 2003; Perrin et al. 2007). If such epimutations affect modifier genes involved in DNA repair, a “slippery slope” of somatic and transgenerational genetic instability (i.e., a mutator phenotype) may result (Jacinto and Esteller 2007), leading not only to an increase in deleterious (purifiable) mutations (Wu et al. 2007; Morak et al. 2008) but also to occasional advantageous (positively selectable) mutations (Sniegowski et al. 2000; Cirz and Romesberg 2007) and/or speciation events (Sniegowski 1998). Selection of such “driver” beneficial mutations may lead in turn to “hitchhiking” of mutator (epi)mutations in modifier genes (Johnson 1999) as “passengers” (Frohling et al. 2007). Such mutational buffering could enhance evolvability (Wagner 2008)—consistent with the idea that error-free DNA repair may be maladaptive in mutagenic or stressful environments (Breivik and Gaudernack 2004; Ponder et al. 2005; Siegl-Cachedenier et al. 2007)—yet may also impair performance and hence robustness (Lenski et al. 2006; Frank 2007; Petrie and Roberts 2007). The “evo-devo” conundrum thus remains as to whether evolvability is indeed selectable (Hendrikse et al. 2007; Lynch 2007), and if so, by what mechanism (Colegrave and Collins 2008; Pigliucci 2008). Even if such a mechanism exists (King and Jukes 1969; Monk 1995), traditional thinking predicts that such selection may act only very weakly at a “good-for-the-species” level (Hiraiwa-Hasegawa 2000).

We have addressed this dilemma by comparing 2 classes of human genes implicated in prevention of cancer, a disease of disordered microevolution (Gatenby and Vincent 2003; Iwasa et al. 2004; Breivik 2005). Tumorigenesis is potentiated by genomic instability (Schneider and Kulesz-Martin 2004; Bielas et al. 2006) arising via multistep inactivation of so-called tumor suppressor genes (Nowak et al. 2004), which, like proto-oncogenes, have been reported to be under strong negative selection pressure (Thomas et al. 2003). These carcinogenic loss-of-function events mainly affect DNA repair—mediated by caretaker genes (CTs) such as BRCA1 and MLH1—or apoptosis, mediated by gatekeeper genes (GKs) such as TP53 and Rb (Kinzler and Vogelstein 1997). These suppressor gene subsets, as well as their disease-causing mutations (Futreal et al. 2004; University Medical Center Groningen 2006), are distinguishable using gene databases (Doctor et al. 2003; Wood et al. 2005). Although long regarded as recessive oncogenes that require 2 “hits” for disease expression (Knudson 2000), suppressor genes are increasingly recognized to exhibit clonal haploinsufficiency in tumors (Santarosa and Ashworth 2004; Smilenov 2006). Given recent evidence for the role of adaptive evolution in cancer progression (Babenko et al. 2006; Crespi and Summers 2006), the occurrence of such haploinsufficiency supports the view that gene loss and pseudogenization (“less is more”) can accelerate genome evolution in certain contexts (Olson 1999). Because deleterious mutations (those causing genetic death) are purged by negative selection, whereas nondeleterious mutations may be positively selected, systematic comparison of CT and GK evolutionary rates should clarify whether these repair and apoptosis gene subsets are subject to distinct evolutionary forces. Consistent with this possibility, comparisons of human and chimpanzee genomes have confirmed different evolutionary rates in functionally distinct gene categories related to tumorigenesis (Clark et al. 2003; Bustamante et al. 2005; Nielsen et al. 2005; Kelley et al. 2006; Voight et al. 2006), whereas adaptive evolution of the BRCA1 CT has been well documented (Huttley et al. 2000; Fleming et al. 2003; Pavlicek et al. 2004). Here, we use genomic data mining to test the hypothesis that germ line CTs are commoner targets for methylation-dependent mutational inactivation than are GKs and, hence, that repair gene dysfunction contributes both to germ line evolvability and somatic tumor progression. A male-dependent prezygotic mechanism for this process, which we have termed programmed genetic instability or PGI (Epstein and Zhao 2006a), is also presented.

The central finding of this study is that CTs are evolving more rapidly than GKs and that this process—which appears likely to be mediated by methylation-dependent mutation—is confined to higher sexually reproducing species. These CT–GK distinctions are consistent with earlier work showing that apoptosis-regulatory genes are essential for development of higher organisms (Aravind et al. 2001), whereas germ line mutation of DNA repair genes drives species evolution (Fay et al. 2001). However, because loss of DNA repair capacity also contributes to cancer development (Cleaver et al. 1995; Breivik and Gaudernack 2004), it is also reasonable to expect tumor suppressor genes in general to be under strong purifying selection (Thomas et al. 2003). Here, we propose that this apparent discrepancy arises from a complex mix of dualistic variables: 1) the bifunctional evolutionary role of repair genes in either conserving genetic information or permitting genetic variation, depending on selection pressure; 2) the bifunctional ability of sexual reproduction either to promote (prezygotically) variation through repair inhibition and intra- or intermale sperm competition or to conserve (postzygotically) genetic fidelity through repair activation, apoptosis, and miscarriage; and 3) the bifunctional role of CpG dinucleotides and promoter CpG islands in either enhancing transcription and repair (when demethylated) or in repressing transcription and predisposing to mutation (when methylated).

Epigenetic reprogramming occurs not only in the early embryo, where somatic patterns of gene expression are set, but also during germ cell development (Morgan et al. 2005)—which changes can be heritable for at least 2 generations (Anway et al. 2008). The methylation dynamics of sperm/testis DNA are unique (Oakes et al. 2007a); unlike oocyte DNA, prezygotic protamine-compacted male germ cell DNA tends to be methylated in nonpromoter regions (Oakes et al. 2007b) that undergo rapid demethylation following fertilization (Haaf 2006). Such sex-specific DNA methylation appears necessary but not sufficient (El-Maarri et al. 1998) to explain the higher mutation rate of mammalian male germ cells (Agulnik et al. 1997; Hurst and Ellegren 1998; Wyckoff et al. 2000; Makova and Li 2002)—suggesting in turn that exposures of the (external) testes to heat (Nikolopoulos et al. 2007), DNA damage (Barber et al. 2006), or other insults (Hara et al. 1999) could well play an evolutionarily programmed epimutagenic role, consistent with the postnatal timing of male germ cell promoter methylation (Driscoll and Migeon 1990). Notably, this hypothesis differs from the standard view of male-driven evolution reflecting a simple excess of male germ cell divisions, giving rise in turn to more replicative mutations (Drost and Lee 1995).

In earlier work, we showed that rarely transcribed genes with promoter CpG islands are hot spots for adaptive evolution (Tang and Epstein 2007), raising the possibility that promoter methylation of sperm target gene classes such as CTs could be a mechanistic “missing link” between environmental selection for specific transcriptomes (Su et al. 2004) and/or coding sequence CpG mutations that permit transgenerational propagation of genetic instability (Wu et al. 2007; Morak et al. 2008). Consistent with this, CTs more often contain promoter CpG islands than do GKs (Zhao Y, unpublished data): classic GKs such as TP53 do not contain promoter CpG islands, whereas canonical CTs such as MLH1 and BRCA1 do. Instructively, the latter gene is mutated and not methylated in familial cancer syndromes (Chen et al. 2006), yet is methylated and not mutated in chemotherapy-induced second malignancies (Scardocci et al. 2006); similar exclusivity between repair gene methylation events and oncogenic indels or point mutations in repair-deficient tumors (Esteller et al. 2001; Toyooka et al. 2006) suggests the fluidity of such epimutations. Although in this model extrinsic damage is the major regulator of prezygotic male germ cell CT methylation—as well as being both a cause and effect of progressive suppressor gene repression in precancerous adult somatic tissues (Neri et al. 2007)—age may be as important as damage in the latter process (Kim et al. 2005), with parental (especially paternal) age playing a synergistic role in the former (Oakes et al. 2003).

We define this model of a “methylation clock” regulating the epigenetic inactivation of CT/repair genes in the male germ line and adult somatic tissues—commensurate with extrinsic damage/stress or intrinsic ageing/senescence—as PGI; just as programmed cell death (PCD) is the mechanism of negative selection, so is PGI proposed to be the mechanism of positive selection (fig. 6). We suggest that PGI intensifies genetic divergence during sexual reproduction at the level of sperm–egg fusion, in contrast to PCD which eliminates both oocyte (Suh et al. 2006) and sperm DNA defects after syngamy (Fatehi et al. 2006). This sequence (sperm → ovum, zygote → embryo) of positive followed by negative selection fits with the notion of sexual conflict (Partridge and Hurst 1998), which should enhance biological system robustness and evolvability (Kitano 2004). We also note that heritable, though not necessarily familial, predisposition to carcinogenesis could also be propagated transgenerationally by PGI (Epstein and Zhao 2006b).

What evidence do we have for PGI operating through the male germ line? The original hypothesis arose from prior knowledge, namely, 1) evidence for male-driven evolution from other groups (Agulnik et al. 1997; Hurst and Ellegren 1998; Wyckoff et al. 2000; Makova and Li 2002); 2) evidence for repair genes being speciation genes, implying a permissive role in rapid evolution (Radman and Wagner 1993; Cleaver et al. 1995; Sniegowski 1998); and 3) the external anatomic location of the testis, making male germ cells uniquely vulnerable to mutagenic, yet potentially reparable, environmental damage (Roest et al. 1996; Hsia et al. 2003; Feitsma et al. 2007). Against this background, we needed to integrate the following new data: 1) CT/repair genes are evolving with unexpected rapidity; 2) CT knockouts are relatively dispensable for survival, resulting only in reduced male fertility; and 3) abnormally low CT/repair gene expression is characteristic of teratozoospermia (Zhao Y, unpublished data), a condition of abnormal sperm morphology that does not significantly reduce in vitro fertilization success rates (Keegan et al. 2007). These considerations invited the hypothesis: could the rapid evolution of CT/repair genes reflect selection for a permissive (i.e., causal loss-of-function) role in male-driven evolution? This latter possibility is supported by the finding that heterozygous males and homozygous females can remain fertile when homozygous male repair gene knockouts are sterile (Roest 1996) and that even minimal repair transgene expression suffices to rescue fertility in repair-knockout males (Hsia 2003). Moreover, homozygous repair-deficient males may survive—albeit at the cost of tumor susceptibility—in genetic backgrounds where homozygous females die (Cranston et al. 1997). This striking gender-specific difference strongly suggests a male-specific significance for accelerated CT evolution, which involves interaction of ambient damage with selection for subtle (e.g., epimutational) male germ cell repair defects.

Our gene targeting and familial cancer data confirm that germ line CT defects are less lethal than GK defects (tables 2 and 3). The effects of repair gene targeting depend critically on the extent of functional inhibition and the presence or otherwise of associated mutations; in general, mild (e.g., haploinsufficient) CT/repair defects tend to increase mutation while decreasing apoptosis (Frank et al. 2005; Smilenov et al. 2005) and may even enhance fitness and lifespan (Siegl-Cachedenier et al. 2007), whereas severe repair defects tend to cause increased apoptosis and premature lethality (Henrie et al. 2003). Hence, because low-level sperm DNA damage can be repaired after fertilization of repair-proficient oocytes (Menezo 2006; Marchetti et al. 2007; Fernandez-Gonzalez et al. 2008)—particularly in the setting of prior DNA damage “conditioning” of such oocytes (Agrawal and Wang 2008)—mild nondeletional prezygotic male germ line repair defects could plausibly enhance sperm divergence with minimal fertility compromise, thereby safeguarding species’ evolvability while offsetting transgenerational risks of paternally transmitted birth defects (Marchetti and Wyrobek 2008) or cancer (Zenzes et al. 1999; Yauk et al. 2007). This conclusion is further supported by the finding that chronic exposure of spermatogonia to low-dose damage greatly reduces genetic damage induced by an acute second hit (Cai et al. 1993; Koana et al. 2007). Our findings therefore suggest the evolution of an environmentally interactive genetic program for promoting divergence in the germ line. We note that the related age-dependent predisposition to cancer is not wholly disadvantageous to the species as such mortality may promote redistribution of environmental resources to younger and more fertile individuals.

As an extension of the “immortal strand” hypothesis of stem cell fidelity (Cairns 2006), our findings raise the notion that the transcribed (well repaired, CpG-demethylated) DNA strand represents the “immovable object” of negative selection, whereas the untranscribed (poorly repaired, CpG-methylated) strand provides the “irresistible force” of positive selection. Furthermore, consistent with the notion that sexual conflict selects for distinct gender phenotypes (Hosken et al. 2001), the evolutionary paradigm presented here implies that the usual targets of positive selection—choice and specialization (Barkman 2003; Hamm et al. 2007), discrimination and success (Swanson et al. 2003), and beauty (Morris RD and Morris JA 2004)—are intrinsically male (sperm/PGI dependent) in origin, whereas the targets of negative selection (normality, utility, longevity, and fidelity) are genetically female or oocyte/PCD dependent. Both positively and negatively selected traits are vital for fitness in sexually reproducing species: for example, the reproductive success of butterflies, birds, and peacocks depends on highly variegated yet symmetric surface patterns (Gould JL and Gould CG 1997) that predict a low underlying frequency of deleterious functional mutations (Morris RD and Morris JA 2004). In our model, PGI is driven by sexual selection pressure on the male germ line to optimize as opposed to normalize whatever phenotypes can be optimized—such as sperm velocity (Gage et al. 2004) or ion channel function regulating motility (Podlaha et al. 2005)—while also enhancing variation in species-discriminatory phenotypes such as egg-binding proteins (Moller and Cuervo 2003). Hence, the basis for sexual conflict in our model is that “average” can never be “best” and that “health” may not guarantee “popularity” (Zaidel et al. 2005)—whether with respect to beauty (DeBruine et al. 2007) or to sperm competition (Neff and Pitcher 2005)—thus helping to explain the paradox that mutation rates tend to be reduced by natural selection yet increased by sexual selection (Moller and Cuervo 2003).

In conclusion, we have shown that CTs are less essential for germ line viability and hence more rapidly evolving than GKs. The resulting model of PGI implicates sexual reproduction as an evolutionary masterstroke: uniquely, sex plays off prezygotic positive selection (in which epimutational divergence is accelerated by environmental stress and damage, and efficiently selected and fixed by intra- and/or intermale sperm competition) against the evolutionary safety valves of postzygotic repair and negative selection (Menezo 2006). Via this mechanism, we propose that environmental stressors succeed in the otherwise oxymoronic task of “selecting for divergence” via epigenetic inhibition of DNA repair, thus helping to settle the “4-billion-year struggle of selfish genes to balance the need for variation with the equally important goal of conserving success” (Gould JL and Gould CG 1997). Moreover, if tumors do indeed arise in part as a side effect of PGI, cancer susceptibility may be most accurately viewed as the tumor-permissive “price” paid by multicellular organisms for genetic plasticity, with the species reaping the ultimate evolutionary “reward” of a delayed time to extinction.

Supplement 1, 2, and 3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).