by Nick Lane
If the success of a fertilized egg depends at least in part on being properly furnished with resources, one might naively imagine that natural selection would favour an equal input from both parents, minimizing the cost to the parents, while maximizing the benefit to offspring. By this measure, sperm add next to nothing, beyond their genes, to the next generation, and so ‘ought’ to be selected against. In fact, they behave like parasites, giving nothing and gaining everything. While parasitic behaviour might plausibly develop in many cases, why are sperm always parasitic? In the case of amphibians and fish, whose eggs are free-floating, the answer might be that millions of tiny sperm can fertilize more eggs, by virtue of their ‘blanket’ coverage. It seems peculiar, though, that the sperm and eggs have retained their extreme size discrepancy even when fertilization is internal. Now, millions of tiny sperm are targeted at one or two eggs closeted in a fallopian tube, rather than thousands of eggs dispersed in the ocean. Is it just too late to change, or not worth bothering; or is there a more fundamental reason for an extreme size discrepancy? There’s a strong argument to say there is.
Uniparental inheritance
The search for a fundamental difference between the sexes takes us back to primitive eukaryotic organisms, such as algae and fungi, some of which have two sexes despite there being no obvious distinctions between their gametes (sex cells). They are said to be isogamous, meaning that their sex cells are equally sized. In fact, the two sexes appear to be identical in every way. Because they are basically the same, it makes more sense to refer to them as mating types rather than sexes. But the very lack of differences between the two mating types highlights the fact that there are still two of them. Individuals are restricted to mating with only half the population. As the pioneers of this field, Laurence Hurst and William Hamilton, pointed out, if finding a mate presents a problem then halving the population size ought to be a serious constraint. Imagine that a mutant mating type appeared in the population, which was able to mate with both the existing mating types. This third type ought to spread swiftly, as it has twice the choice of mates. Any subsequent mutants that could mate with all three types would have a similar advantage. The number of mating types should therefore tend towards infinity; and indeed the widespread ‘split gill’ mushroom Schizophyllum commune has 28 000. Short of having no sexes at all (all are the same sex) then it makes sense to have as many as possible. Two is the worst of all possible worlds.
So why do many isogamous species still have two mating types? If there really is a deep asymmetry between the sexes, a grain of inequality from which all other inequalities grow, then the algae and fungi are the place to look.
The answer betrays a fundamental intolerance that makes our own battle of the sexes look like a love-in. Take the primitive alga Vulva, for example, also known as the sea lettuce. It is a multicellular alga, which forms into sheets of cells only two cells thick but up to a metre long, giving the appearance of leaves. Sea lettuces produce identical gametes, or isogametes, which contain both chloroplasts and mitochondria. The two gametes, and their nuclei, fuse together in a perfectly normal fashion, but after cell fusion the organelles attack each other with savage ferocity. Within a couple of hours of fusion, the chloroplasts and mitochondria deriving from one of the gametes have been pulped to a swollen mass, and soon afterwards disintegrate altogether.
This is an extreme example of a general trend. The common denominator is an intolerance of the organelles from one of the two parents, but the method of extermination varies widely. Perhaps the most illuminating example is the single-celled alga Chlamydomonas rheinhardtii, which at first sight appears to buck the trend. Instead of destroying half its chloroplasts in a wanton display of violence, the chloroplasts fuse together peaceably. But biochemical scrutiny shows this alga is no more tolerant than its cousins; rather, it is more refined in its intolerance, like a cultivated Nazi. To use the correct, rather chilling euphemism, Chlamydomonas practices ‘selective silencing’: it eliminates the DNA in the organelles rather than the whole organelles, leaving the infrastructure intact. The organelle DNA from each parent attacks the other with lethal DNA-digesting enzymes. According to some reports, 95 per cent of all the organelle DNA is dissolved, but the speed of destruction is slightly faster on one side than the other. The surviving DNA, by definition, derives from the ‘maternal’ parent.
The upshot is that nuclear fusion and recombination are fine, but the organelles—the chloroplasts and mitochondria—are almost invariably inherited from only one parent. The problem is not with the organelles, but with their DNA. There’s something about this DNA that fate abhors. Two cells fuse, but only one of them passes on the organelle DNA.
Here lies the deepest difference between the sexes: the female sex passes on organelles, the male sex does not. The result is uniparental inheritance, which means that organelles, such as mitochondria, are normally inherited only down the maternal line, like Judaism. The realization that mitochondria are inherited only from the mother is not age-old knowledge: it was first reported in 1974, in horse-donkey hybrids, by the geneticist and jazz pianist Clyde Hutchison III and his colleagues at the University of North Carolina.
Is this really the deepest difference between sexes? The best place to look for a reality check is at any apparent exceptions to the rule. We already noted, for example, that the mushroom S. commune has 28 000 mating types. These are encoded by two ‘incompatibility’ genes on different chromosomes, each of which comes in many possible versions (alleles). An individual inherits one out of more than 300 possible alleles on one chromosome, and one out of more than 90 on the other, giving a total of 28 000 possible combinations. If two cells share the same allele on either chromosome, they cannot mate. This is likely to be the case among siblings, which encourages out-breeding. However, if the gametes have different alleles at both loci, they are free to mate, and this allows them to mate with more than 99 per cent of the population, rather than a feeble 50 per cent like the rest of us.
But with all these sexes, how on earth do the fungi keep track of their organelles? Can they, too, ensure that organelles are inherited from only one of two parents? If so, with 28 000 sexes, how do they know which is the ‘mother’? In fact they solve the problem by engaging in a fastidious form of sex, a loveless fungal missionary position, and are zealous never to mix bodily fluids. Sex, for S. commune, is all about getting two nuclei into the same cell, and the cytoplasm never conjoins in blissful union: cell fusion does not take place. In other words, these fungi get around the sex problem altogether by evading the issue. While it can be said that they have 28 000 different sexes, it is better to say they don’t have any at all: they have incompatibility types instead.
Intriguingly, incompatibility types can coexist with sexes in the same individual, implying that these adaptations really do serve different functions. The best examples come from the flowering plants, or angiosperms, many of which, as we have seen, are hermaphrodites (the individuals are both male and female). In principle, this means that plants can fertilize themselves or their closest relations—and in practice, given the difficulties of dispersal faced by the sessile plants, this would be the most likely scenario. The trouble is that local fertilization favours in-breeding, thereby losing the benefits of sex altogether. Many angiosperms get around the problem by having incompatibility types as well as two sexes, ensuring that out-breeding takes place.
In principle, it’s possible to have more than two sexes, while maintaining uniparental inheritance. There are examples of this among primitive eukaryotes, notably the slime moulds, which fuse cells together into a matrix with numerous nuclei sharing the same vast cell. Slime moulds look similar to fungi, overgrowing woody mulch or grass as an amorphous mass; some bright yellow moulds have been likened to dog vomit. From our point of view, the most important aspect is that some of them have more than two sexes, even though the whole gametes fuse together, not just the nuclei. The best known example is Physarum polycephalum, which has a
t least 13 sexes, encoded by different alleles of a gene known as matA. While looking the same, however, these sexes are not equal—their mitochondrial DNA is ranked in a pecking order. Upon fusion of the gametes, the mitochondrial DNA of the more dominant strain persists, while that of the subordinate strain is digested, and disappears completely within a couple of hours; the vacant sheaths are eliminated within three days of fusion. So uniparental inheritance is preserved despite the occurrence of multiple sexes. Presumably there is a limit to how high a pecking order can rise; it’s hard to imagine a hierarchy accommodating all 28 000 sexes of S. commune, for example. And in practice, more than two sexes is rare.
To draw a general conclusion, we can say that the act of sex involves nuclear fusion (and out-breeding can be enforced by having incompatibility types), but proper sexes can only be distinguished when cytoplasm is shared. In other words, sexes develop when the cells as well as their nuclei fuse. Then the female passes on some of her organelles, and the male must accept the untimely demise of all of his. Even when there are multiple sexes, uniparental inheritance of the mitochondria is the rule.
Selfish competition
Why is uniparental inheritance so important? And why are multiple sexes so uncommon, given that they expand the mating opportunities and are technically feasible? The most widely accepted reason was developed as a forceful hypothesis by Leda Cosmides and John Tooby, at Harvard, in 1981. They argued that mixing the cytoplasm from two different cells creates an opportunity for conflict between different cytoplasmic genomes. These include both mitochondrial and chloroplast genomes, but also any other cytoplasmic ‘passengers’ such as viruses, bacteria, endosymbionts, and so on. If these passengers are genetically identical, there can be no competition between them; but as soon as they begin to differ, there is scope for competition to gain entrance to the gametes.
Consider, for example, two different populations of mitochondria, one of which replicates faster than the other. If one population becomes more numerous, it gains preferential entry to the gametes. The other population will be eliminated unless it speeds up its own replication, and to do so almost certainly means that it will fail to do its proper job, generating energy, as effectively. This is because the easiest way to speed up replication is to jettison ‘unnecessary’ genes, as we saw in Part 3; and the genes that are unnecessary for mitochondrial replication are of course exactly the genes that the cell as a whole needs for energy production. So competition between mitochondrial genomes leads to an evolutionary arms race, in which selfish interests take precedence over the interests of the host cell.
The host cell inevitably suffers from competition between mitochondrial genomes, and this in turn generates a strong selective pressure on the genes in the nucleus to ensure that all the mitochondria are identical, thereby preventing such conflict. This can be achieved by the ‘selective silencing’ of one population, as in Chlamydomonas, but in general it is safest to preclude their entrance altogether; this simultaneously precludes competition between other cytoplasmic elements, such as bacteria and viruses. Thus, in this selfish theory, the reason that two sexes develop is because this is the most effective means of preventing conflict between selfish cytoplasmic genomes.
The male mitochondria don’t take their purging lying down. Any attempt to exclude them is met with stiff resistance. The angiosperms attest eloquently to the reality of selfish mitochondrial behaviour. In these hermaphrodite flowering plants the mitochondria strive to avoid being caged in the male part of the plant, a dead end for them because they are not passed on in pollen. They avoid ending up in pollen by sterilizing the male sex organs, usually by bringing about the abortion of pollen development. Not surprisingly, this is an important trait in agriculture, discussed at length by Darwin himself, and known rather forbiddingly as male cytoplasmic sterility. By sterilizing the male sex organs, the mitochondria convert a hermaphrodite into a female, thereby helping safeguard their own transmission. However, because this upsets the sex balance of the population as a whole, which now comprises females and hermaphrodites, various nuclear genes that counteract the selfish mitochondrial actions have been selected over evolution, restoring full fertility. The battle is still being waged. A trail of selfish mitochondrial mutants and nuclear suppressor genes shows that female conversion took place repeatedly, only to be suppressed each time. In Europe today, 7.5 per cent of angiosperm species are gynodioecious, to use the term Darwin coined—their population comprises both females and hermaphrodites.
Hermaphrodites are particularly vulnerable to male sterilization, because the female organs leave open the possibility of mitochondrial transmission in the same individual. But even when the male and female sex organs are housed in separate individuals, there are indications that mitochondria attempt to distort the sex balance by harming males. Some diseases, notably Leigh’s hereditary optic neuropathy, are caused by mutations in the mitochondrial DNA, and are more prevalent in men than women. This situation is similar to the action of Wolbachia in arthropods, which we noted earlier. In crustaceans, infection with Wolbachia converts males into females, but in many insects the effect is even more drastic: males are simply killed. The ‘objective’ of the bacteria, which are passed on from one generation to the next only in the egg, is to convert the entire population into females, thereby improving their own chances of transmission. Mitochondria, too, can help safeguard their own transmission in the egg by eliminating males. Unlike Wolbachia, however, their success seems to be very limited. Presumably, this is because there has been a stronger counter-selection against selfish mitochondria. Fully functional mitochondria are essential to our survival and health, and selfish mutants are less likely to be effective at respiring. They are therefore likely to be heavily selected against. Wolbachia, in contrast, distorts the sex ratio but doesn’t necessarily cause much damage otherwise; accordingly, there is a lower selective pressure against it.
All these various attempts to subvert the sex ratio come about because mitochondria, along with other cytoplasmic elements such as chloroplasts and Wolbachia, are passed on only in the egg. The pressure to bend the rules has almost certainly polarized the existing differences between the sperm and eggs even further. For example, the pressure exerted by selfish mitochondria probably contributed to the extreme size difference between sperm and eggs. The simplest way to tip the scales against the selfish mitochondria is to stack the odds against them. There are some 100 000 mitochondria in human egg cells, but fewer than 100 in sperm. If the male mitochondria get into the egg at all (they do in many species, including ourselves), they are simply diluted out. But even dilution is not enough. Numerous tricks have evolved to exclude male mitochondria from the fertilized egg altogether, or to ensure the permanent silencing of the few that do get in. In mice and humans, for example, the male mitochondria are tagged with a protein called ubiquitin, which marks them up for destruction in the egg. In most cases, the male mitochondria are degraded within a few days of entry to the egg. In other species the male mitochondria are excluded from the egg altogether, or even from the sperm, as in crayfish and some plants.
Perhaps the most bizarre method of excluding the male mitochondria is found in the giant sperm of some species of fruit fly (Drosophila), which can be more than ten times longer than the total male body length when uncoiled. The testes required to produce such mammoth sperm comprise more than 10 per cent of the total adult body mass, and retard male development markedly. Their evolutionary purpose is unknown. Such extraordinary sperm add far more cytoplasm to the egg than normal. What’s more, the sperm tail persists in the egg, raising the question of its fate. According to Scott Pitnick and Timothy Karr, at Syracuse University, New York, and the University of Chicago, respectively, the mitochondria fuse together during sperm development to form two enormous mitochondria, which extend the entire length of the tail. These two vast mitochondria fill 50 to 90 per cent of the total cell volume. They are not digested in the egg, but rather are sequestered throughout embr
yonic development, ultimately in the midgut. The sperm tail can still be observed in the midgut of the larvae after hatching, and is defaecated out soon after birth, conforming to the spirit of uniparental inheritance, if in an oddly convoluted manner.
The fact that there are so many radically different methods of excluding the male mitochondria implies that uniparental inheritance evolved repeatedly, in response to similar selection pressures. This in turn suggests that uniparental inheritance was also lost repeatedly, and later regained by way of whatever trick was most readily pressed into service at the time. I suspect this means that losing uniparental inheritance was weakening but rarely fatal, and indeed there are some examples of mitochondrial mixing, or heteroplasmy, notably among the fungi and angiosperms. For example, in one large study of 295 angiosperm species, nearly 20 per cent of all the species examined showed some degree of bi-parental inheritance. Interestingly, bats, too, are often heteroplasmic. Bats are long-lived, vigorously active mammals, so it is curious that they are not compromised by heteroplasmy. Little is known about the circumstances or selection pressures involved, but there is a hint that some sort of selection for the fittest mitochondria might take place in the flight muscles themselves.
We have brought mitochondrial heteroplasmy upon ourselves in some assisted reproductive technologies, especially ooplasmic transfer. This technique involves the injection of cytoplasm, along with its mitochondria, from a healthy donor egg into the egg cell of an infertile woman, thereby mixing mitochondria from two different women. We touched on this technique in the Introduction, for it found fame in a newspaper under the headline ‘Babies born with two mothers and one father.’ More than thirty apparently healthy babies have been born by this method, despite the pungent criticism that it ‘may be akin to trying to improve a bottle of spoiled milk by adding a cup of fresh.’ The profound disquiet felt about mixing two mitochondrial populations, which nature strives so hard to avoid, combined with the suspiciously high rate of developmental abnormalities leading to miscarriage, has led to the technique being placed on hold in the United States. Even so, to an open-minded sceptic, perhaps the most surprising finding is that it works at all. Certainly heteroplasmy is worrying, probably weakening, but not out of the question.