Nuclear versus organelle

The offspring of sexually reproducing organisms inherit approximately half of their DNA from each parent. In a diploid, sexually reproducing organism for example, this means that within the nuclear genome one allele at each locus came from the mother and the other allele came from the father. This is known as biparental inheritance. However, even in sexually reproducing species, not all DNA is inherited from both parents. Two important exceptions are the unipar-entally inherited organelle genomes of mitochondria (mtDNA) and plastids, with the latter including chloroplasts (cpDNA). These are both located outside the cell nucleus. Mitochondria are found in both plants and animals, whereas plastids are found only in plants. Organelle DNA typically occurs in the form of supercoiled circles of double-stranded DNA, and these genomes are much smaller than the nuclear genome. For example, at between 15 000 and 17 000 bp the mammalian mitochondrial genome is approximately 1/10 000 the size of the smallest animal nuclear genome, but what they lack in size they partially make up for in number -a single human cell normally contains anywhere from 1 000 to 10 000 mitochondria. Molecular markers from organelle genomes, particularly animal mtDNA, have been exceedingly popular in ecological studies because, as we shall see below, they have a number of useful attributes that are not found in nuclear genomes.

Animal mitochondrial DNA

Mitochondrial DNA is involved primarily in cellular respiration, the process by which energy is extracted from food. Animal mtDNA contains 13 protein-coding genes, 22 transfer RNAs and two ribosomal RNAs. There is also a control region that contains sites for replication and transcription initiation. Most of the sequences are unique, i.e. they are non-repetitive, and there is little evidence of either spacer sequences between genes or intervening sequences within transcribed genes. Although some rearrangement of mitochondrial genes has been found in different animal species, the overall structure, size and arrangement of genes are relatively conserved (Figure 2.1). In most animals, mitochondrial DNA is inherited maternally, meaning that it is passed down from mothers to their offspring (although there are exceptions; see Box 2.1).

There are several reasons why mtDNA markers have been used extensively in studies of animal population genetics. First of all, mtDNA is relatively easy to work with. Its small size, coupled with the conserved arrangement of genes, means that many pairs of universal primers will amplify regions of the mitochondria in a wide variety of vertebrates and invertebrates. This means that data often can be obtained without any a priori knowledge about a particular species' mitochondrial DNA sequence. Second, although the arrangement of genes is conserved, the overall mutation rate is high. The rate of synonymous substitutions in mammalian mtDNA

Control region

12S rRNA

Control region

12S rRNA

Figure 2.1 Typical gene organization of vertebrate mtDNA. Unlabelled dark bands represent 22 transfer RNAs (tRNAs). Gene abbreviations starting with ND are subunits of NADH dehydrogenase, and those starting with CO are subunits of cytochrome c oxidase

Figure 2.1 Typical gene organization of vertebrate mtDNA. Unlabelled dark bands represent 22 transfer RNAs (tRNAs). Gene abbreviations starting with ND are subunits of NADH dehydrogenase, and those starting with CO are subunits of cytochrome c oxidase has been estimated at 5.7 x 10~8 substitutions per site per year (Brown et al., 1982), which is around ten times the average rate of synonymous substitutions in protein-coding nuclear genes. The non-coding control region, which includes the displacement (D) loop, evolves particularly rapidly in many taxa. The high mutation rate in mtDNA may be due partly to the by-products of metabolic respiration and also to less-stringent repair mechanisms compared with those acting on nuclear DNA (Wilson et al., 1985). Regardless of the cause, these high mutation rates mean that mtDNA generally shows relatively high levels of polymorphism and therefore will often reveal multiple genetic lineages both within and among populations.

The third relevant property of mtDNA is its general lack of recombination, which means that offspring usually will have (barring mutation) exactly the same mitochondrial genome as the mother. As a result, mtDNA is effectively a single haplotype that is transmitted from mothers to their offspring. This means that mitochondrial lineages can be identified in a much more straightforward manner than nuclear lineages, which, in sexually reproducing species, are continuously pooling genes from two individuals and undergoing recombination. The effectively clonal inheritance of mtDNA means that individual lineages can be tracked over time and space with relative ease, and this is why, as we will see in Chapter 5, mtDNA sequences are commonly used in studies of phylogeography.

Finally, because mtDNA is haploid and uniparentally inherited, it is effectively a quarter of the population size of diploid nuclear DNA. Because there are fewer copies of mtDNA to start with, it is relatively sensitive to demographic events such as bottlenecks. These occur when the size of a population is temporarily reduced, e.g. following a disease outbreak or a catastrophic event. Even if the population recovers quickly, it will have relatively few surviving mitochondrial haplotypes compared with nuclear genotypes. As we will see in the next chapter, inferring past bottlenecks can make an important contribution towards understanding the current genetic make-up of populations.

Box 2.1 Mitochondrial DNA: exceptions to the rules

Uniparental inheritance and a lack of recombination have made mtDNA the molecular marker of choice in many studies of animal populations because these properties mean that, until a mutation occurs, all of the descendants of a single female will share the same mitochondrial haplo-type. This means that genetic lineages can be retraced through time using relatively straightforward models. However, as with so many things in biology, there are exceptions to the rules of mitochondrial inheritance. For one thing, not all mitochondria in animals are inherited maternally. Instances of paternal leakage (transmission of mitochondria from father to offspring) have been found in a number of species, including mice (Gyllensten et al., 1991), birds (Kvist et al., 2003) and humans (Schwartz and Vissing, 2002). Nevertheless, the extent of paternal leakage is believed to be low in most animals with the exception of certain mussel species within the families Mytilidae, Veneridae and Unionidae, which follow double biparental inheritance. This means that females generally inherit their mitochondria from their mothers, but males inherit both maternal and paternal mtDNA. The males therefore represent a classic case of heteroplasmy (more than one type of mitochondria within a single individual).

Bivalves by no means represent the only group in which heteroplasmy has been documented, but its prevalence in some mussel species makes them an ideal taxonomic group in which to investigate another question that has been posed recently: do animal mitochondria sometimes undergo recombination? The lack of identifiable recombinant mtDNA haplotypes in natural populations has led to the assumption that no recombination was taking place, but there was always the possibility that recombination was occurring but was remaining undetected because it involved two identical haplotypes. The presence of more than one mtDNA haplotype in male mussels meant that recombination could, at least in theory, produce a novel haplotype, and this indeed has proved to be the case (Ladoukakis and Zouros, 2001; Burzynski et al., 2003). These findings suggest that mtDNA recombination may be more common than was previously believed in the animal kingdom, although at the moment there is little evidence that recombination, heteroplasmy or paternal leakage are compromising those ecological studies that are based on mtDNA sequences.

Plant mitochondrial DNA

As with animals, mtDNA in most higher plants is maternally inherited. There are a few exceptions to these rules, for example mtDNA is transmitted paternally in the redwood tree Sequoia sempervirens and biparentally inherited in some plants in the genus Pelargonium (Metzlaff, Borner and Hagemann, 1981). The overall function of plant and animal mitochondria is similar but their structures differ markedly. Unlike animal mtDNA, plant mitochondrial genomes regularly undergo recombination and therefore evolve rapidly with respect to gene rearrangements and duplications. As a result, their sizes vary considerably (40 000 - 2 500 000 bp). This variability makes it difficult to generalize; to take one example, the mitochondrial genome of the liverwort Marchantia polymorpha is around 186 608 bp long and appears to include three ribosomal RNA genes, 29 transfer RNA genes, 30 protein-coding genes with known functions and around 32 genes of unknown function (Palmer, 1991). Although the organization of plant mitochondria regularly changes, evolution is slow with respect to nucleotide substitutions. In fact, in most plant species the mitochondria are the slowest evolving genomes (Wolfe, Li and Shorg, 1987). The overall rates of nucleotide substitutions in plant mtDNA are up to 100 times slower than those found in animal mitochondria (Palmer and Herbon, 1988), and this low mutation rate combined with an elevated recombination rate means that plant mitochondrial genomes have not featured prominently in studies of molecular ecology.

That is not to say that there are no useful applications of mtDNA data in plant studies. For many plant species, dispersal is possible through either seeds or pollen, which often vary markedly in the distances over which they can travel. For example, if seeds are eaten by small mammals they may travel relatively short distances before being deposited. In contrast, pollen may be dispersed by the wind, in which case it could travel a long way from its natal site. Even if seeds are wind dispersed they are heavier than pollen and therefore still likely to travel shorter distances. Nevertheless, it is also possible that the opposite scenario could occur, for example seeds that are ingested by migratory birds may travel much further than wind-blown pollen. Tracking seeds and pollen is extremely difficult, but the different dispersal abilities of the two sometimes can be inferred by comparing the distributions of mitochondrial and nuclear genes. Because mtDNA is usually inherited maternally, its distribution will reflect the patterns of seed dispersal but will not be influenced by the spread of pollen, which contains only the paternal genotype.

Canadian populations of the black spruce (Picea mariana) grow in areas that were covered in ice until approximately 6000 years ago, so we know that populations must have been established since that time. Researchers found that current populations share the same mtDNA haplotypes but not the same nuclear alleles (Gamache et al., 2003). This difference was attributed to the widespread dispersal of nuclear genes that were carried by wind-blown pollen, coupled with a much more restricted dispersal of mitochondrial genes that can be transported only in seeds. Because seeds usually do not travel very far, it is likely that only a few were involved in establishing populations once the ice had retreated, hence the lack of variability in mtDNA. On the other hand, the pollen that blew to these sites most likely originated in multiple populations, and this has led to a much higher diversity in nuclear genotypes. If this study had been based solely on data from either nuclear or mitochondrial DNA we would have an incomplete, and possibly misleading, picture denoting the dispersal of this coniferous species.

Plastids, including chloroplast DNA

The relatively low variability of plant mtDNA means that when haploid markers are desirable in plant studies, researchers more commonly turn to plastid genomes, including chloroplast DNA (cpDNA). Like mtDNA, cpDNA is inherited maternally in most angiosperms (flowering plants), although in most gymnosperms (conifers and cycads) it is usually inherited paternally. Chloroplast genomes, which in most plants are key to the process of photosynthesis, typically range from 120 000 to 220 000 bp (the average size is around 150 000 bp). Although recombination sometimes occurs, chloroplasts are for the most part structurally stable, and most of the size variation can be attributed to differences in the lengths of sequence repeats, as opposed to the gene rearrangement and duplication found in plant mtDNA.

In tobacco (Nicotiana tabacum), the cpDNA genome contains approximately 113 genes, which include 21 ribosomal proteins, 4 ribosomal RNAs, 30 transfer RNAs, 29 genes that are necessary for functions associated with photosynthesis and 11 genes that are involved with chlororespiration (Sugiura, 1992). A partial arrangement of chloroplast genes in the liverwort (Marchantia polymorpha) is shown in Figure 2.2. The average rate of synonymous substitutions in the chloroplast genome, at least in higher plants, is estimated as nearly three times higher than that in plant mtDNA (Wolfe, Li and Sharp, 1987), although this is still four to five times slower than the estimated overall rate of synonymous substitutions in plant nuclear genomes (Wolfe, Sharp and Li, 1989). However, this is an average mutation rate, and the use of cpDNA markers in plant population genetic studies has escalated in recent years following the discovery of highly variable microsatellite regions within the chloroplast genome (see below).

23S rRNA

23S rRNA

16S rRNA

Figure 2.2 The genome of the chloroplasts found in the liverwort Marchantía polymorpha contains 121 024 base pairs (Ohyama et al, 1986). These make up an estimated 128 genes, and the approximate locations of some of these are shown on this figure. The dark lines mark the locations of 12 of the 37 tRNAs

16S rRNA A

16S rRNA

Subunits of RNA polymerase

Rubisco (large subunit)

Figure 2.2 The genome of the chloroplasts found in the liverwort Marchantía polymorpha contains 121 024 base pairs (Ohyama et al, 1986). These make up an estimated 128 genes, and the approximate locations of some of these are shown on this figure. The dark lines mark the locations of 12 of the 37 tRNAs

Even when variability is low, genetic data from chloroplasts continue to play an important role in ecological studies, in part because of their uniparental mode of inheritance. In the previous section, we saw how a comparison of data from mtDNA (dispersed only in seeds) and nuclear DNA (dispersed in both seeds and pollen) provided insight into the relative contributions that seeds and pollen make to the dispersal patterns of black spruce. Another way to approach this question in conifers, in which chloroplasts are inherited paternally, is to compare the distribution of chloroplast genes (dispersed in both seeds and pollen) with the distribution of mitochondrial genes (dispersed only in seeds). Latta and Mitton (1997) followed this approach in a study of Limber pine (Pinus flexilis James) in Colorado. The seeds of this species are dispersed by Clark's nutcracker (Nucifraga columbiana), which caches seeds within a limited radius of the natal tree. If these caches are subsequently abandoned, they may grow into seedlings. Pollen, on the other hand, is dispersed by the wind, and therefore should travel further than the seeds. As expected, the mitochondrial haplotypes of Limber pine were distributed over much smaller areas than the chloroplast haplotypes, once again supporting the notion of relatively widespread pollen dispersal (Latta and Mitton, 1997).

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