Mutation and recombination

Genetic variation is created by two processes: mutation and recombination. Most mutations occur during DNA replication, when the sequence of a DNA molecule is used as a template to create new DNA or RNA sequences. Neither reproduction nor gene expression could occur without replication, and therefore its importance cannot be overstated. During replication, the hydrogen bonds that join the two strands in the parent DNA duplex are broken, thereby creating two separate strands that act as templates along which new DNA strands can be synthesized. The mechanics of replication are complicated by the fact that the synthesis of new strands can occur only in the 5'-3' direction (Figure 1.5). Synthesis requires an enzyme known as DNA polymerase, which adds single nucleotides along the template strand in the order necessary to create a complementary sequence in which G is paired with C, and A is paired with T (or U in RNA). Successive nucleotides are added until the process is complete, by which time a single parent

Replication fork

Replication fork

Figure 1.5 During DNA replication, nucleotides are added one at a time to the strand that grows in a 5' to 3' direction. In eukaryotes, replication is bi-directional and can be initiated at multiple sites by a primer (a short segment of DNA)

DNA duplex (double-stranded segment) has been replaced by two newly synthesized daughter duplexes.

Errors in DNA replication can lead to nucleotide substitutions if one nucleotide is replaced with another. These can be of two types: transitions, which involve changes between either purines (A and G) or pyrimidines (C and T); and transversions, in which a purine is replaced by a pyrimidine or vice versa. Generally speaking, transitions are much more common than transversions. When a substitution does not change the amino acid that is coded for, it is known as a synonymous substitution, i.e. the DNA sequence has been altered but the encoded product remains the same. Alternatively, non-synonymous substitution occurs when a nucleotide substitution creates a codon that specifies a different amino acid, in which case the function of that stretch of DNA may be altered. Although single nucleotide changes often will have no phenotypic outcome, they can at times be highly significant. Sickle-cell anaemia in humans is the result of a single base-pair change that replaces a glutamic acid with a valine, a mutation that is generally fatal in homozygous individuals.

Errors in DNA replication also include nucleotide insertions or deletions (collectively known as indels), which occur when one or more nucleotides are added to, or removed from, a sequence. If an indel occurs in a coding region it will often shift the reading frame of all subsequent codons, in which case it is known as a frameshift mutation. When this happens, the gene sequence is usually rendered dysfunctional. Mutations can also involve slipped-strand mis-pairing, which sometimes occurs during replication if the daughter strand of DNA temporarily becomes dissociated from the template strand. If this occurs in a region of a repetitive sequence such as a microsatellite repeat, the daughter strand may lose its place and re-anneal to the 'wrong' repeat. As a result, the completed daughter strand will be either longer or shorter than the parent strand because it contains a different number of repeats (Hancock, 1999).

Mutations are by no means restricted to one or a few nucleotides. Gene conversion occurs when genotypic ratios differ from those expected under Mendelian inheritance, an aberration that results when one allele at a locus apparently converts the other allele into a form like itself. In the 1940s, Barbara McClintock discovered another example of gene alterations called transposable elements, which are sequences that can move to one of several places within the genome. Not only are these particular elements relocated, but they may take with them one or more adjacent genes, resulting in a relatively large-scale rearrangement of genes within or between chromosomes. Transposable elements can interrupt function when they are inserted into the middles of other genes and can also replicate so that their transposition may include an increase in their copy number throughout the genome. Many also are capable of moving from one species to another following a process called horizontal transfer, a possibility that is being investigated by some researchers interested in the potential hazards associated with genetically modified foods.

The other key process that frequently alters DNA sequences is recombination. Most individuals start life as a single cell, and this cell and its derivatives must replicate many times during the growth and development of an organism. This type of replication is known as mitosis, and involves the duplication of an individual's entire complement of chromosomes -- in other words the daughter cells contain exactly the same number and type of chromosomes as the parental cells. Mitosis occurs regularly within somatic (non-reproductive) cells.

Although necessary for normal body growth, mitosis would cause difficulties if it were used to generate reproductive cells. Sexual reproduction typically involves the fusion of an egg and a sperm to create an embryo. If the egg and the sperm were produced by mitosis then they would each have the full complement of chromosomes that were present in each parent, and the fused embryo would have twice as many chromosomes. This number would double in each generation, rapidly leading to an unsustainable amount of DNA in each individual. This is circumvented by meiosis, a means of cellular replication that is found only in germ cells (cells that give rise to eggs, sperm, ovules, pollen and spores). In diploid species (Box 1.1), meiosis leads to gametes that have only one set of chromosomes (n), and when these fuse they create a diploid (2n) embryo. During meiosis, recombination occurs when homologous chromosomes exchange genetic material. This leads to novel combinations of genes along a single chromosome (Figure 1.6) and is an important contributor to genetic diversity in sexually reproducing taxa.

Locus 1 I Locus 2 | Locus 3

Locus 1 I Locus 2 \ Locus 3

Allele a j Al!e!e B Allele c

Figure 1.6 Recombination at the gene level, after which the gene sequence at chromosome 1 changes from ABC to AbC Recombination often involves only part of a gene, which typically leads to the generation of unique alleles

Box 1.1 Chromosomes and polyploidy

The karyotype (the complement of chromosomes in a somatic cell) of many species includes both autosomes, which usually have the same complement and arrangement of genes in both sexes, and sex chromosomes. The number of copies of the full set of chromosomes determines an individual's ploidy. Diploid species have two sets of chromosomes (2n), and if they reproduce sexually then one complete set of chromosomes will be inherited from each parent. Humans are diploid and have 22 pairs of autosomes and two sex chromosomes (either two X chromosomes in a female or one X and one Y chromosome in a male), which means that their karyotype is 2n = 46. Polyploid organisms have more than two complete sets of chromosomes. The creation of new polyploids sometimes results in the formation of new species, although a single species can comprise multiple races, or cytotypes. In autopolyploid individuals, all chromosomes originated from a single ancestral species after chromosomes failed to separate during meiosis. In this way, a diploid individual (2n) can give rise to a tetraploid individual (4n), which would have four copies of the original set of chromosomes. This contrasts with allopoly-ploid individuals, which have chromosomes that originated from multiple species following hybridization.

Polyploidy is very common in flowering plants and also occurs to a lesser degree in fungi, vertebrates (primarily fishes, reptiles and amphibians) and invertebrates (including insects and crustaceans). Polyploidy is of ecological interest for a number of reasons, for example newly formed polyploids may either outcompete their diploid parents or co-exist with them by exploiting an alternative habitat. Habitat differentiation among cytotypes of the same species has been documented in a number of plant species. Ecological differences between cytotypes also may depend on unrelated species, for example tetraploid individuals of the alumroot plant Heuchera grossulariifolia living in the

Rocky Mountains are more likely than their diploid conspecifics to be consumed by the moth Greya politella, even when the two cytotypes are living together (Nuismer and Thompson, 2001). There will be other examples throughout this text that show the relevance of ploidy to molecular ecology.

Neutralists and selectionists

Prior to the 1960s, most biologists believed that a genetic mutation would either increase or decrease an individual's fitness and therefore mutations were maintained within a population as a result of natural selection (the selectionist point of view). However, many people felt that this theory became less plausible following the discovery of the high levels of genetic diversity in natural populations that were revealed by allozyme data in the 1960s, since there was no obvious reason why natural selection should maintain so many different genotypes within a population. At this time the Neutral Theory of Molecular Evolution began to take shape (Kimura, 1968). This proposed that although some mutations confer a selective advantage or disadvantage, most are neutral or nearly so, that is to say they have no or little effect on an organism's fitness. The majority of genetic polymorphisms therefore arise by chance and are maintained or lost as a result of random processes (the neutralist point of view). For a while, reconciliation between selectionists and neutralists seemed unlikely, but the copious amount of genetic data that we now have access to suggests that molecular change can be attributed to both random and selective processes. As a result, many well-supported theories of molecular evolution and population genetics now embrace elements of both neutralist and selectionist theories (Li and Graur, 1991).

There are a number of predictions that can be made about mutation rates under the neutral theory. For example, synonymous substitutions should accumulate much more rapidly than non-synonymous substitutions because they are far less likely to cause phenotypic changes. In general, this prediction has been borne out. Data from 32 Drosophila genes revealed an average synonymous substitution rate of 15.6 substitutions per site per 109 years compared with an average non-synonymous substitution rate of 1.91; similarly, the synonymous substitution rate averaged across various mammalian protein-coding genes was 3.51 compared with a rate of 0.74 in non-synonymous substitutions (Li, 1997, and references therein). As we may expect under the neutral theory, mutations tend to accumulate more rapidly in introns (non-coding regions) compared with exons (non-coding regions), and pseudogenes appear to have higher substitution rates compared with functional genes, although this conclusion is based on limited data (Li, 1997).

A combination of chance and natural selection means that a proportion of mutations will inevitably be maintained within a species and this accumulation of mutations, along with recombination, means that even members of the same species often have fairly divergent genomes. Overall, around 0.1 per cent of the human genome (approximately three million nucleotides) is variable (Li and Sadler, 1991), compared with around 0.67 per cent of the rice (Oryza sativa) genome (Yu et al., 2002). In molecular ecology, studies are typically based on multiple individuals from one or more populations of the species in question, and overall levels of sequence variability are usually expected to be around 0.2 - 0.5 per cent (Fu and Li, 1999), although this may be considerably higher depending on the gene regions that are compared. Sequence divergence also tends to be higher between more distantly related groups, and therefore comparisons of populations, species, genera and familes will often show increasingly disparate genomes, although there are exceptions to this rule (Figure 1.7). Part of the challenge to finding suitable genetic markers for ecological research involves

Comparison between harbour seal and grey seal Comparison between fin whale and blue whale Comparison between mouse and rat

Pairwise comparisons of 18 different mitochondrial regions

Figure 1.7 Sequence divergence based on pairwise comparisons of 18 different randomly numbered regions of mtDNA for members of two different genera from the same family (harbour seal Phoca vitulina and grey seal Halichoerus grypus); members of the same genus (fin whale Balaenoptera physalus and blue whale B. musculus); and members of two different families (mouse Mus musculus and rat Rattus norvegicus). As we might expect, sequence divergences are highest in the comparison between families (mouse and rat). However, contrary to what we might expect, the congeneric whale species are genetically less similar to one another than are the two seal genera. This is an example of how taxonomic relationships do not always provide a useful guide to overall genetic similarities. Data from Lopez et al. (1997) and references therein identifying which regions of the genome have levels of variability that are appropriate to the questions being asked.

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