Animal mitochondrial DNA as a genetic marker in population and evolutionary biology

TREE vol. 4, no. 7, January

plant population genetics and plant breeding was made obvious. However, it was plant breeding in a prerecombinant DNA era, with emphasis on composite cross populations and a belief that natural selection might produce a balanced population of genotypes which, in terms of disease resistance and yield, would surpass that of specific varieties produced by man. In reality,

this never happened. It remains true that an understanding of the population genetics of a species is fundamental to efficient germplasm conservation, which in turn will continue to underpin crop improvement even in the current era of recombinant DNA technology. Mainly due to Allard and his associates, approaches to investigating the population genetics of plants are

AnimalM itochondrial DNAasa GeneticMarkerin Population and Evolutionary Biology RichardG. Harrison Animal mitockondrial DNA (&DNA) is playing an iflcreasingly important role us a genetic marker in population and evolutionary biology. The popularity of this molecule derives, in part, from the relative euse with which clearly homologous sequences can be isolated and compared. Simple sequence organization, maternal inheritance and absence of recombinatioti make mtDNA an ideal marker for tracing maternal genealogies. Rapid rate of sequence divergence (at least in vertebrates) allows discrimination of recently diverged lineages. Studies of mtDNAs from u diversity of animal groups have revealed significant variation among taxa in mtDNA sequence dynamics, gene order and genome size. They have also provided import& insights into population structure, geographic variation, zoogeography and phylogeny.

The ability to assess DNA sequence divergence among individuals, populations, species or higher taxa has revolutionized systematic and evolutionary biology. Currently available techniques differ in the nature of the information they provide and in the proportion of the genome that can be assayed. Complete sequencing of homologous DNA fragments from different the most organisms provides powerful and direct method for obtaining information on amount of Richard Harrison is at the Section of Ecology and Systematics,Corson Hall, Cornell University, Ithaca. NY 14853,USA.

6

genetic variation or extent of genetic divergence. Each nucleotide base pair represents a character that can exist in four discrete character states, and the number of characters is effectively limited only by the time and money available for sequence analysis. Use of the polymerase chain reaction (PCR) to amplify homologous DNA sequences from many individuals or taxa should lead to a rapid expansion of the DNA sequence data base. At the other end of the spectrum, DNA-DNA hybridization gives a single estimate of genetic distance averaged over the entire genome. This approach may avoid problems that arise from examining only a small portion of the genome, but it is limited in other ways (e.g. it does not produce discrete character state data). information about mtDNA variation in natural populations has come principally from comparisons of restriction enzyme fragment patterns and site mapsi-4. Restriction endonucleases cleave doublestranded DNA at specific recognition sequences, producing a series of fragments, the number and sizes of which vary depending on where within the DNA the recognition sequence occurs. Thus fragment pattern differences can conveniently serve as markers of genetically distinct lineages. Simple measures of genetic distance can be derived from com-

1989

now well defined. In future, such approaches will be further integrated with ecology and widely applied in studies of plant population biology.

References 1 Allard, R.W. (1960) Principles ofPlant Breeding, Wiley 2 Brown,A.H.D. andAllard,R.W. (1970) Genetics66,133-145

parisons of restriction site maps or restriction fragment patterns. Alternatively, each site (or fragment) can be treated as a character having two states (present or absent), thereby providing a data set appropriate for phylogenetic analysis5. Characteristics of animal mitochondrial DNA The small, closed-circular mitochondrial genomes of animals are clearly homologous DNA sequences3,6. Since pure samples of mtDNA can be prepared from small amounts of tissue with relative ease, it is straightforward to compare homologous mtDNA sequences from a wide variety of organisms. Although total nuclear DNA is easy to prepare, isolating hon-4ogous nuclear sequences is more difficult and has typically necessitated constructing and screening genomic libraries for each individual or species involved. (Selective amplification using PCR provides an important new shortcut.) Gene content and genome organization Animal mtDNA exhibits remarkable conservation of gene content. MtDNA molecules from vertebrates, insects and echinoderms include two ribosomal RNA (rRNAI genes, 22 transfer RNA (tRNA) genes, and I3 genes that code for proteins involved in electron transport or ATP synthesis3.6, (Fig. 1 I. Each mtDNA molecule also has a control region containing sequences that function in initiation of replication and transcription. Gene order is conserved among vertebrates, but comparisons across phyla indicate that major rearrangements have occurred’. The mtDNA molecule in animals is exceptionally compact, with few in-

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tergenic sequences, no introns and, except in the control region, few examples of large insertions or duplications. MtDNA in fungi and plants is larger in size and more complex in genome organization. The size of the mtDNA molecule in animals varies within and between species, ranging from about 14 kilobases (kb) to more than 30 kb (Refs 3 and 6). Fig. I. Gene content

Transmission genetics Animal mtDNA is maternally inherited. In crosses between strains of moths, mice or fish that differ in mtDNA genotype, multiple generations of backcrossing of females to males of the paternal parental type has revealed no evidence of a paternal contribution7-9. Maternal inheritance has important consequences. Because only a fraction of the population (half if the sex ratio is 1 : 1) pass on their mtDNA to offspring, the effective population size for mtDNA is smaller than that for nuclear genes. Consequently, stochastic processes will be particularly important in determining frequencies of mtDNA Genetic affinities genotypeslo,“. defined by mtDNA genotypes reflect matriarchal phylogeny. MtDNA should, therefore, be an excellent tracer of patterns of colonization, including founder events. Gene flow mediated by male dispersal will not affect spatial patterns of variation for mtDNA. Comparisons of patterns of variation for mtDNA with those for sequences transmitted by both sexes (autosomal genes) or those transmitted only by males (e.g. Y-linked genes) may reveal differences in male and female population structure and perhaps suggest underlying differences between the sexes in ecology and behavior. Because there are many mtDNA molecules per cell (including the germ cells), females transmit many copies of the mtDNA molecule to each offspring. A new mtDNA variant arises as a mutation in a single molecule within a single cell lineage. The process of fixation of that variant must occur both within the population of molecules in a germ cell lineage and within the population of organisms”,‘*. During at least the initial stages of this process of fixation, cell lineages should exist that contain two types

and arrangement in vertebrate (VI and fruit fly (D) mtDNA. The two taxa have identical gene content, but differ by a number of transpositions and inversions. Vertebrates are represented by human, cow, mouse (Mus) and toad (Xenopus), fruit flies by Drosophila yakuba. Hatched regions are the 22 transfer RNAs. Abbreviations: CR, control region; b, cytochrome b; I-111, subunits of cytochrome oxidase; A6 and A8, subunits of ATP synthetase; l-6 lincl. 4L), subunits of NADH dehydrogenase; sr and Ir, small and large subunits of ribosomal RNA; o and o,,, origins of replication. Note that the molecules are colinear (except for the tRNAsl in the segment between the arrows. Redrawn from Refs 3 and 6.

of mtDNA (the original type and the new variant). Such lineages (individuals) are referred to as heteroplasmic. Despite the presence of considerable restriction site variation among individuals, such variation within individuals (heteroplasmy) is rare. In contrast, heteroplasmy involving length variation is common (except in mammals) (Table II. This difference is presumably a consequence of a higher mutation rate for length variants than for site variantsly. If the waiting time between mutations is long compared with the time to fixation or loss, segregation of mitochondrial genotypes will occur rapidly and most individuals will be homoplasmic. In the extreme case, the effective population size for mitochondrial genes will only be one-fourth that for nuclear genes’ I. RecomGination In animals, recombination between mtDNA molecules occurs rarely or not at aIF. If inheritance is strictly maternal, differentiated genomes are not brought together during sexual reproduction. Even if recombination events do occur, the products of recombination are unlikely to represent new mtDNA genotypes and will not be recognized as recombinants. Evolutionary biologists, therefore, have access to a set of completely linked markers which permit clear definition of maternal genealogies and excellent discrimination beand common ancestry tween convergencelp4. However, the high resolution provided by mtDNA is

offset, in part, by the fact that the entire mtDNA genome represents a single genetic marker. Thus, stochastic events and selection will influence the frequency of entire mtDNA genotypes. Although there is complete linkage of all mtDNA markers, they are unlinked to nuclear genes. When linkage relationships restrict the independence of nuclear gene markers, the behavior of mtDNA is likely to be different from that of nuclear genes and patterns of variation for nuclear and mitochondrial markers may not be concordant (see below). Sequence evolution Studies of mtDNA evolution in primates suggest an initial rapid rate of sequence divergence (0.5I .O% per lineage per million years), 5-10 times the rate observed nuclear DNA for single-copy (scnDNA114n15. Data from a variety of tetrapod groups appear to be consistent with the estimates from primates3,4. However, in flies and sea urchins, mtDNA and scnDNA may evolve at similar rateslG18. It is apparent that there is considerable variability among taxa in relative and absolute rate+. A universal mtDNA molecular clock cannot be assumed. Certainly a careful calibration of the clock for a variety of invertebrate lineages is essential before mtDNA can be used to estimate divergence times for these groups. Rates of sequence evolution vary along the mtDNA molecule. Sequences in the large and small ribosomal RNA genes are highly 7

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Table I. Examplesof mtDNA length variation and heteroplasmy Species”

-

Type of variation and deletions

Locatio+

HeteroplasmyC

Ref.

D-loop and noncoding region

None

37

Homo sapiens

Small additions

Mus musculus

Large deletion

Coding region

Rare - but deletion found only in heteroplasmic condition

38

Cnemidophorus

spp.

Tandem duplications of various sizes (0.8-8.0 kb)

D-loop and adjacent coding region

Rare

39

Cnemidophorus C. tesselatus

tigris /

64 bp repeat

D-loop

23%

40

Hyla cinerea / H. gratiosa

Many size classes

Localized

l-8%

41

Triturus cristatus

Many large insertions

D-loop

Rare

42

Rana esculenta

Continuous

D-loop

100%

43

Amia calva

Discrete size classes

Localized

8%

41

220 bp tandem

A+T-rich

>20%

13,44

Many discrete size classes

A+T-rich

18%

45

470 bp tandem

A+T-rich

20%

46

Localized

7%

47

Gryllus pennsylvanicus G. firmus Drosophila

melanogaster

D. melanogaster Placopecten

/

subgroup

magellanicus

variation

1.2 kb tandem

aMus, mice; Cnemidophorus, lizards; Hyla, frogs; fruit flies; Placopecten, scallops. bRegion of the mtDNA molecule and insect mtDNA, respectively. “This column

gives the percentage

in which

of

sequence

repeat repeat Triturus,

size variation

of individuals

conserved, whereas base substitutions and rearrangements accumulate rapidly in the control region3,6. Because of this intramolecular variability, sequence comparisons of different regions of the molecule can provide useful information for taxa at very different levels of divergence. For recently diverged taxa, base substitutions occur primarily in intergenic sequences, in the control region, or at sites within coding regions that do not result in amino acid replacement3,6. Rates of nucleotide substitution appear to slow down when these sites have been saturated, presumably because there are strong functional constraints on tRNA and rRNA sequences and on the proteins encoded by mtDNA. Estimates of mtDNA sequence divergence among populations, species and genera come primarily from comparisons of restriction fragment patterns or restriction site maps. Across all taxa, there is no clear correspondence between the extent

repeat

divergence

newts;

Rana, frogs; Amia, bowfins

occurs. The D-loop and A+T-rich

(fish); Gryllus, field crickets;

regions

are the control

regions

Drosophila, in vertebrate

that carry more than one size class of mtDNA.

and the taxonomic rank of the populations being compared. For example, conspecific sunfish (genus Lepomisl may have mtDNA molecules that differ in sequence by as much as &IO% (Ref. 191, whereas comparisons between birds placed in different genera (Melospiza, Zonotrichial reveal only 6% sequence divergencezo. Mitochondrial DNA as a genetic marker Population structure Because mtDNA exhibits considerable variation among individuals both within and between populations, it has proved to be an effective marker of population structure and patterns of intraspecific geographic variationz. In several recent studies, analysis of the distribution of mtDNA genotypes has provided evidence for the geographical structuring of populations*. In the pocket gopher Ceomys, the horseshoe crab Limu/us. and a number of freshwater fishes in the southeastern United

States,

distinct

mtDNA

lineages

oc-

cupy different parts of the species’ ranges. In each case, there appears to be a maior discontinuity. presumably reflecting current environmental and/or historical influences. In contrast, analysis of mtDNA genotypes in populations of the American eel Anguilla reveals no geographical differentiation, consistent with the suggestion that eels throughout eastern North America represent a single panmictic population. Population bottlenetlis and colonization events Maternal inheritance and absence of recombination make mtDNA a particularly appropriate marker for tracing recent evolutionary history, including colonization (founder) events, introductions and population bottlenecksd. Data on mtDNA variation in human populations have been especially revealing. Early studies uncovered relatively little variation, leading to the suggestion of a recent popu-

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lation bottleneck21. Assuming a constant rate of mtDNA sequence divergence, estimates of existing amounts of divergence can be used to calculate the time since two individuals (species) last shared a common female ancestor. The amount of variation in human populations, revealed by recent comparisons of detailed restriction site maps for 147 humans, suggests that a common female ancestor of all modern humans lived about 200000 years ago22. This assumes a rate of divergence of l-2% per lineage per million years. The implication is not that only a single human female existed at that time, but that all living humans have derived their mtDNA (but not necessarily their nuclear alleles) from one female. Although the date of the common female ancestor is surprisingly recent, the persistence of only one maternal lineage from that time is easily explained by random lineage extinction. Lineage sorting in finite populations will cause the diversity

of mtDNA lineages to decrease over time, at a rate considerably faster than for single nuclear gene markerslo. As a simple illustration, it is evident that females who have only sons will not contribute any mtDNA to future generations, although their nuclear genes will be passed on. Thus, it is not necessary to invoke a severe bottleneck to explain observed levels of mtDNA variation in human populations. The human mtDNA restriction maps also provide insights into historical patterns of migration and colonization. Relationships among the mtDNA genotypes of the 147 individuals are consistent with an African origin for all modern humans and suggest that each nonAfrican population is the result of multiple colonization eventsz2. The mtDNA data do not appear to support the hypothesis that over the past million years modern humans have evolved in parallel in several parts of the world. Other recent studies have also

focused on geographic origins and patterns of colonization, relying on analysis of the geographic distribution of mtDNA genotypes determined from restriction site maps or fragment patterns. Data for Drosophila simulans support the hypothesis that the Indo-Pacific is the original home of this species, which has probably attained a worldwide distribution only recently23. Analyses of mtDNA polymorphisms in Columbian ground squirrels in Canada and deer mice on the California Channel Islands and adjacent mainland provide evidence for both the times and routes of Pleistocene or postPleistocene colonization events24e25. Natural 6y6ridization events In various groups of animals, interspecific hybridization can result in parthenogenetic or hybridogenetic offspring. Although comparison of nuclear gene markers can be used to identify the two parental species, mtDNA analysis allows

TREE vol. 4, no. I, January

00

l

0

0 0

Oo

O

.o 0

0

0 0

00

00

l

0

O 0

. 0

(a>

000c3 O 0 L-J 0

0

n 0

0

0

00 00 0

0 0

.

QIl (CY Fig. 2. A scenario

for random lineage sorting of mtDNA (or other genetic marker) within local populations derived from a polymorphic ancestor. Lineage sorting can result in lack of concordance between mtDNA relationships and species boundaries. For example, a large polymorphic population (al is subdivided into local demes tbl. Following subdivision. each small population becomes fixed for one of the two mtDNA genotypes (represented by open or filled symbols). In (d), one population has become sufficiently differentiated (in morphology, ecology, behavior, etc.1 to be called a distinct species and is, therefore, represented by squares rather than circles. This species has a mtDNA genotype identical to that found in one of the populations of its more widespread sister species. The latter is clearly paraphyletic with respect to mtDNA.

unambiguous identification of the maternal parent3. Similarly, mtDNA analysis, in concert with allozyme data, can reveal asymmetries in mate choice or reproductive isolation between hybridizing taxa. Such asymmetries should lead, in theory, to disequilibria between mtDNA genotypes and nuclear gene markers and result in asymmetric introgression of mtDNA26. Empirical support for these predictions has come from studies of tree frogs in which there is an asymmetry in mating behavior*’ and of field crickets in which post-mating barriers to gene exchange are present in only one of the two reciprocal crosses28. Genetic affinities and mutriarclral Phvbwnv

Perhaps the most widespread use of mtDNA as a marker has been

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to define genetic affinities among closely related species based on comparisons of restriction fragment patterns or restriction site maps. There is no clear consensus about methods of phylogenetic inference from such datai. Questions have arisen about the reliability of comparisons, fragment pattern the appropriateness of distance measures, problems of homoplasy (since sites have only two statespresent and absent), and the relative merits of different parsimony methods (since there are multiple ways to lose a restriction site but only one way to create a new site). has Nevertheless, mtDNA already proved to be a useful marker for defining relationships among closely related species. Data from restriction site maps and fragment pattern comparisons have been used to examine relationships in a variety of genera (e.g. Peromyscus29, MusSO, Drosophi/a’6.31 1. In many cases, relationships defined on the basis of mtDNA comparisons are concordant with relationships defined by morphological, behavioral or allozyme characters. However, there are also a surprising number of examples of discordance - e.g. greater similarity in mtDNA genotype between individuals in different species than between at least some pairs of conspecific individuals. Two classes of explanation have been invoked to explain the apparent lack of concordance between mtDNA variation and species boundaries defined on the basis of other characters. In cases in which species are known to hybridize, differential introgression of mtDNA may explain the presence in one species of a mtDNA genotype similar or identical to one of the mtDNA genotypes found in a second species (Table 2). Hybrid zone theory suggests that patterns of introgression for a neutral marker will depend on the linkage relationships of the marker to genes that contribute to reproductive isolation32. Close linkage to genes that contribute to reproductive isolation (e.g. hybrid unfitness or positive assortative mating) will significantly retard the flow of a neutral marker allele across a hybrid zone. Since mtDNA is unlinked to the nuclear

genome, the extent of introgression of a mtDNA variant will be greater than for many or most nuclear markers (assuming that many loci contribute to reproductive isolation133. If genes contributing to reproductive isolation are few in number, many nuclear markers may be effectively unlinked to this set of genes and will flow across a hybrid zone at the same rate as mtDNA. In at least some hybrid zones, differential introgression has occurred (Table 21 and in the case of European house mice, Scandinavian populations of Mus musculus have become fixed for a mtDNA genotype characteristic of its close relative, Mus domesticd4. In other hybrid zones, patterns of variation for mtDNA and nuclear gene markers are completely concordant (e.g. in hybridizing European toads in the genus Bombina)35. Lack of concordance between mtDNA and nuclear gene markers can also result from the random extinction of lineages in populations (species) derived from a polymorphic ancestor29,3(). For example, consider a widespread species polymorphic for mtDNA genotypes A and B. Isolation of local populations, followed by random extinction of the A or B lineage at each site, can lead to a situation in which overall genetic similarity of populations is not necessarily correlated with their mtDNA similarity (Fig. 21. In the extreme case, one subpopulation might become sufficiently differentiated to be recognized as a separate species and yet, by chance, may share mtDNA genotypes with a subset of the remaining populations. In such a situation, the widespread species will appear to be paraphyletic with respect to mtDNA genotype. This appears to be the case for Peromyscus maniculatus and P. polionotus29. Models of lineage sorting during speciation predict that species may initially appear polyphyletic or paraphyletic. These arguments are not limited to comparisons between mitochondrial and nuclear gene markers. Phylogenies based on different nuclear genes may not be concordant - and differential introgression and lineage sorting can again be invoked to explain observed discrepancies. Allele phylogenies are not

TREE vol. 4, no. 1, January

1989

equivalent to organism PhYlogenies, and to the extent that alleles behave independently, species boundaries and species relationships may need to be defined gene by gene32. Understanding the factors that control the association of mtDNA genotype with nuclear genes is of great importance to evolutionary and systematic biologists, since it is the nuclear genes and their products that determine most aspects of an organism’s phenotype. MtDNA serves as a marker of recent maternal ancestry and its utility depends on a predictable association between mitochondrial and nuclear genotypes. This association is clearly affected by stochastic events and by differential introgression, and may also be influenced by natural selection. Since the products of mtDNA genes closely interact with nuclear gene products, coevolution of mtDNA and nuclear sequences may occur3.

ing and differential introgression can lead to discordance between patterns of mtDNA variation and those seen for nuclear gene markers. Recent studies of mtDNA from a large number of different taxa have challenged the notions that size variation and heteroplasmy are rare and that mtDNA always evolves at a faster rate than scnDNA. Clearly, properties of mtDNA defined for one group of organisms cannot be assumed to be properties of the molecule in other groups. This conclusion is inconvenient but important.

Conclusions Analyses of mtDNA sequence divergence can provide detailed information about phylogenetic relationships at several levels in the evolutionary hierarchy. Because of maternal inheritance and the absence of recombination, the mtDNA molecule is an ideal marker for tracing maternal genealogy. It can also serve as an extremely useful diagnostic tool for differentiating conspecific populations, e.g. in identifying the source population of a recent introduction or range expansion. MtDNA is potentially a useful marker over a considerable phylogenetic expanse, both because rates of sequence divergence are not constant along the molecule and because the several approaches to assessing sequence divergence differ greatly in their resolving power. Recent studies of population structure, natural hybridization events, phylogenetic relationships and regional zoogeography attest to the utility of mtDNA analysis in documenting patterns of variation within and between species. However, patterns of mtDNA variation must be interpreted with some caution. Since mtDNA represents a single genetic marker unlinked to the nuclear genome, both random lineage sort-

References

Acknowledgements I am grateful to Chip Aquadro, Thorn Boyce, Tim Collins, David Rand, Carol Yoon and four anonymous reviewers for providing comments on an earlier draft of this paper. My research involving mtDNA has been supported by NSF Grant BSR-8407474 and by several grants from USDA.

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