DNA Fingerprints from Hypervariable Mitochondrial Genotypes' John ...

DNA Fingerprints from Hypervariable Mitochondrial Genotypes’ John C. Avise,* Brian W. Bowen,* and Trip Lamb t *Department

of Genetics, University of Georgia; and tsavannah

River Ecology Laboratory

Conventional surveys of restriction-fragment polymorphisms in mitochondrial DNA of menhaden fish (Brevoortia tyrannus/patronus complex) and chuckwalla lizards (Sauromalus obesus) revealed exceptionally high levels of genetic variation, attributable to differences in mtDNA size as well as in restriction sites. The observed probabilities that any two randomly drawn individuals differed detectably in mtDNA genotype were 0.998 and 0.983 in the two species, respectively. Thus, the variable gel profiles provided unique mtDNA “fingerprints” for most conspecific animals assayed. mtDNA fingerprints differ from nuclear DNA fingerprints in several empirical respects and should find special application in the genetic assessment of maternity. Introduction

The term DNA jngerprinting has come to be associated with a molecular approach introduced by Jeffreys et al. (19853, 1985c) in which Southern blot assays of hypervariable regions of DNA are employed to reveal genotypes that are often individual specific. The hybridization probes employed by Jeffreys et al. are conserved core sequences within tandem-repetitive regions of DNA (minisatellites) dispersed in the nuclear genome. The hypervariability reflected in the Southern blot gel profiles results from unequal exchanges during meiosis that alter the number of tandem repeats at the numerous minisatellite “loci.” This capacity to distinguish with heritable markers most or all individuals in outbred species has found applications in forensic medicine (Dodd 1985; Gill et al. 1985); paternity and maternity testing (Jeffreys et al. 1985a; Burke and Bruford 1987), linkage analysis (Jeffreys and Morton 1987; Jeffreys et al. 1987), and demographic genetics (Hill 1987; Wetton et al. 1987; Hoelzel and Amos 1988). Any region of DNA sufficiently polymorphic to provide individual-diagnostic markers should allow similar opportunities for genetic fingerprinting. For example, a sequence in the bacteriophage M 13 hybridizes to another class of hypervariable minisatellites that may also distinguish individual animals (Vassart et al. 1987); and extensive polymorphisms at loci influencing histocompatibility can be revealed as rejection responses in tissue grafts between genetically nonidentical individuals (Angus and Schultz 1979; Neigel and Avise 1983; O’Brien et al. 1985). In the present study, we report that some species of vertebrates harbor sufficient polymorphism in mitochondrial DNA (mtDNA) sequence to permit individual-diagnostic genetic fingerprinting by conventional restriction-digestion analysis. Within both the menhaden fish (Clupeidae: Brevoortia tyrannuslpatronus complex) and the 1. Key words: genetic polymorphism, restriction sites, maternity analysis. Address for correspondence and reprints: John C. Avise, Department of Genetics. University of Georgia, Athens, Georgia 30602. Mol. Biol. Evol. 6(3):258-269. 1989. 0 1989 by The University of Chicago. All rights reserved.

0737-4038/89/0603-0004$02.00

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chuckwalla lizard (Iguanidae: Sauromahs obesus), differences in mtDNA sizes and in restriction sites conspire to produce extensive mtDNA polymorphisms which unambiguously distinguish a very high fraction of the conspecific individuals assayed. Here we document these results and use the findings to motivate a consideration of the similarities and distinctions between genetic fingerprinting methods that are based on clonally transmitted (mitochondrial) versus sexually transmitted (nuclear) genes. Material and Methods The hypervariability of mtDNA in menhaden and chuckwallas was uncovered during surveys of molecular zoogeography in several species of marine fishes in the southeastern United States (B. W. Bowen and J. C. Avise, unpublished data) and in desert reptiles in the American Southwest (T. Lamb, T. R. Jones, and J. C. Avise, unpublished data). Brevoortia tyrannus and B. patronus are sister taxa of clupeid fishes inhabiting shallow waters along the Atlantic and Gulf Coasts, respectively. A total of 33 fish were assayed: B. tyrannus, Chesapeake Bay, MD (N=8) and Brunswick, GA (N=9); B.patronus, Ocean Springs, MS (N= 16). Chuckwallas are large iguanid lizards that inhabit the desert Southwest. A total of 5 1 individuals, collected from 13 sites in Arizona (N=23), 11 sites in California (N=26), and one site in Nevada (N=2) were examined. Zoogeographic and systematic ramifications of the mtDNA data sets for the menhaden and chuckwallas will be addressed elsewhere. Here we will confine attention to the diversity of mtDNA genotypes observed and to the significance of this in the context of DNA fingerprinting. From the fresh heart, liver, and/or gonadal tissue of each individual, mtDNA was purified by standard procedures involving cesium-chloride gradient centrifugation (Lansman et al. 1981). Restriction digestions were conducted overnight under conditions recommended by the enzyme suppliers. mtDNAs from the menhaden were digested with the following informative restriction enzymes (those producing at least two cuts in some mtDNA genotypes): AvaI, AvaII, BcZI, BgZI, BgZII, BstEII, CZaI, EcoRI, EcoRV, HincII, HindIII, KpnI, NdeI, PstI, PvuII, SacI, SpeI, and SM. A similar array of informative enzymes was used to assay the chuckwallas, although CZaI,EcoRV, KpnI, NdeI, PstI, PvuII, and Sac1 were excluded (for reasons of difficulties in scoring or because of lack of production of multiple cuts in the mtDNA). The resulting mtDNA fragments were end labeled with 35-S-radionuclides, separated through 1.O%- 1.6% agarose gels, and revealed by autoradiography (Brown 1980; Maniatis et al. 1982). Fragment sizes were estimated by comparison to a 1-kb molecular-weight standard purchased from Bethesda Research Laboratories. Molecular heterogeneity due to restriction-site variation was distinguished from that due to variation in mtDNA size by procedures detailed in the Results section. Digestion profiles distinguished by site differences were designated by various uppercase letters; variants due to size differences were denoted by primes following the appropriate letter designations. Restriction sites were labeled with lowercase letters. Estimates of nucleotide sequence divergence, calculated by the “site” approach of Nei and Li ( 1979), were based solely on the fragment profile differences attributable to restriction-site changes. Values of genotypic diversity (the “nucleon diversity” of Nei and Tajima 198 l), calculated as n( 1 - Cf f)/n - 1, where fi: is the frequency of the ith mtDNA genotype in a sample of size n, were based on the total numbers of genotypes as identified by both size and site differences. Genotypic diversity can range from 0, when all individuals exhibit the same genotype, to 1.O, when each individual is unique.

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Table 1 Number of Distinct mtDNA Digestion Profiles Produced by Various Endonucleases in Menhaden Fishes and Chuckwalla Lizards MENHADEN

Mean (range) No. of Fragments/Profile

ENZYME AVQI AVUII BclI

... . ..

BglII . . . . Bst EII . . . . ChI . . . EcoRI . EcoRV . . . HincII . . . . HindHI. . . . KpnI . NdeI ... PstI . . . . . PVUII . . . Sac1 . . . . . . SpeI . . . . . . St241 . . . . .

.... ....

5.6 (5-6) 11.1 (8-14) 2.0 6.3 (5-8) 2.0 (l-3) 2.2 (l-3) 1.8 (1-2) 2.5 (2-3) 3.3 (I-5) 8.4 (7-10) 2.0 3.6 (3-4) 2.0 3.1 (2-5) 5.2 (3-7) 1.5 (l-2) 6.1 (5-7) 6.0 (5-7)

(N=33)

CHUCKWALLAS

Total No. of Distinct Fragment Profiles 5 20 1 3 4 6 4 2 6 12 1 7 2 7 17 2 8

Mean (range) No. of Fragments/Profile 5.1 10.4 4.9 3.7 1.7 1.8

(4-6) (9-13) (3-6) (3-4) (l-2) (l-2)

1.5’( i-2) 7.8’(;-8) 6.5 (6-7) . . .

(N=51) Total No. of Distinct Fragment Profiles 7 11 9 6 3 4 . . . 2 . . . 6 4

. . . . . . . . . 5.8’(;-?) 13.2 (12-14)

Results

Menhaden Among the 33 Brevoortia assayed by 18 five- and six-base restriction endonucleases, 32 different mtDNA genotypes were observed. In other words, only two individuals (a B. tyrannus from Brunswick, GA, and a B. patronus from Ocean Springs, MS) appeared identical in our mtDNA assays. (For simplicity, the data for B. tyrannus and B. patronus will be considered collectively, since the species are very closely related and are of questionable taxonomic status. If the species were considered separately, all conclusions about mtDNA fingerprinting would still hold-all 17 B. tyrannus had differentiable mtDNA genotypes, as did all 16 B. patronus.) Many of the restriction endonucleases yielded a large number of distinct digestion patterns (table 1). For example, PvuII alone produced a total of 17 profiles (figs. 1,2), 12 of which (A-L; fig. 2) clearly differed by presence versus absence of various restriction sites, while the remainder (F’-J’; fig. 2) probably involved mtDNA size differences. As an illustration, FvuII B differs from PvuII A by a restriction-site gain converting a 4.6kb fragment to two fragments of sizes 3.3 kb and 1.3 kb (fig. 2). On the other hand, PvuII J’appears to differ from PvuII J only in size, as reflected in the 2.85kb fragment (fig. 2). [Note: it is conceivable that this and other presumptive size differences may in reality represent restriction-site changes. For example, a PvuII restriction-site gain could have converted the 2.85kb fragment in J’to 2.8-kb and 0.05-kb fragments in J-the latter fragment would not have been detectable in our assays. The usual method of verifying mtDNA size heterogeneity, by a search for concordance in molecular-

Pvu I I SITE

VARIANTS

SIZE

r

.

A 9.5 -

C

B

9.5 -

9.5-

E

D

H

G

J

I

6.7 -

6.7 ~6.7

-

6 .7 -

4.0 3.3 2 .8 -2.8

3.3 -

. 28-

2.8 -2.4

-

-2.4

2.4-2.4

-

2.4 -2.4

-

13. -

H”

I’

6.7-

J’

6.7-

6.7-

. 52-

-

3.3 2.8 -

3.6 3.3 ;‘,j =

2.4 -

2.4 -

2.4 -2.4

1.6 E 1.3-

151:3-1.3

4.0 3.32.85 -2.65 -2*5 2.0 -

3.33 3-2.85 -

-2.4

F2*5

.

2.8 .3.85

1.51.3-

13.

-

151:3-

1.3-

1.3-

3.3 -

-2.4

-

. -1.5 1.3-1.3

-

B2*5

16-

1.6 13.

H’

5.2 -

5.2 -

3.3-

-7 F’

L

6.7-

4.6 -

2 .4 -2.4

K

8.0 -

6.7 4.6 -

F

VARIANTS

I

0.8 0.6 -

0.6 -

0.6 -

FIG. 2.-Diagrammatic representation of all PvuII digestion profiles observed in menhaden. Numbers are fragment sizes (in kilobases). The fifth largest fragment in pattern K was actually 2.5 kb long (fig. 1) but is shown here as 2.4 kb to emphasize homology with the 2.4-kb fragments observed in the other profiles.

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weight differences across gel profiles, was difficult to apply to the menhaden (and chuckwallas) because of the extreme diversity in fragment positions that was attributable to the mtDNA site variation. Whenever alternative explanations involving size versus site variation arose, we took the conservative approach (in terms of estimating sequence divergence) of provisionally assigning the difference to variation in mtDNA size.] From study of the PvuII digestion profiles, it was possible to deduce restrictionsite maps (fig. 3). Most of the sites could be placed on the map without ambiguity, whereas a few (such as h or j) were placed only at either of two alternative positions. For example, site j in map L is either 0.8 kb from site a (as shown) or 0.8 kb from site i (fig. 3). Such alternatives have no effect on estimation of sequence divergence. The various maps in figure 3 arise from presences versus absences of PvuII recognition sequences at eight of the 10 observed restriction-site positions (a-d and g-j). The mtDNA size differences noted above appear to map to two separate positions on the genome (in the regions between sites e and f and between sites a and i, respectively). mtDNA size variation is common in other species of poikilothermic vertebrates (Densmore et al. 1985; Bermingham et al. 1986), and, in at least one species (Pseudacris crucifer), the size differences are reportedly due to sequences dispersed in the genome (Moritz et al. 1987). For most of the other endonucleases, it was similarly possible to partition the variation among digestion profiles into restriction-site versus probable mtDNA size differences. However, two enzymes (AvaII and H&II) produced gel profiles that were too heterogeneous for such scoring, and we were forced to limit profile scorings to “same” or “different” among any pair of individuals. Although information from these enzymes was not included in the estimates of nucleotide sequence divergence, the profiles were informative in terms of increasing the overall number of mtDNA fingerprints observed. A total of 92 restriction sites (with an average of - 5S/individual) were scored in the menhaden. On the basis of the restriction-site differences, the estimated mean sequence divergence (base substitutions per nucleotide) between the mtDNA genotypes of all assayed menhaden was p = 0.024 (range 0.000-0.063). Genotypic diversity was 0.998, very near the maximum assumable value of 1.0. Chuckwallas The number of distinct digestion patterns produced by the informative endonucleases is summarized in table 1. For example, AvaII and StuI alone each produced 11 different mtDNA profiles. Among the 5 1 chuckwallas assayed, 35 different composite mtDNA genotypes were observed. No more than three individuals shared any mtDNA genotype. As with the menhaden, it proved feasible to partition most of the fragmentpattern variation into mtDNA size differences versus restriction-site gains and losses. Only digestion-profile changes resulting from the latter were used in the sequencedivergence calculations. These calculations included information on a total of 5 1 restriction sites from nine enzymes (with an average of 37 sites scored/individual). Only the profiles produced by AvaII and StuI proved too complex for such site-map interpretations (see fig. 4). For the reasons discussed above, it was sometimes difficult to identify mtDNA size variation unambiguously. Nonetheless, on the basis of concordant differences in genome size estimates across several restriction digests, the mtDNAs of three chuckwallas from Southern California were almost certainly -0.7 kb larger than those of

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Avise et al.

a

a i

e

e

a

e

b

I

i

d

e

e

a

a

i

i

i

d

e

e

a

a

i

i

i

a

i

e

e

f FIG. 3.-PvuII

restriction-site

maps deduced

from the digestion

profiles shown in fig. 2

many other individuals assayed, and that of a fourth individual from Southern California was clearly - 1.O kb larger than the “norm” of - 16.5 kb for the species. On the basis of the composite restriction-site data, the estimated mean sequence divergence between the mtDNA genotypes of all assayed chuckwallas was p = 0.0 15 (range 0.000-0.028). Genotypic diversity was 0.983.

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Discussion

Nearly all menhaden assayed (and a majority of the chuckwallas) could be distinguished by conventional mtDNA restriction analyses. Among 33 menhaden and 51 chuckwallas, 32 and 35 distinct mtDNA genotypes were observed, respectively. The proximate reasons for the mtDNA hypervariability involve extensive restrictionsite polymorphism, superimposed on mtDNA size differences. The evolutionary explanations for this hypervariability are less clear. Under a neutral model with given mutation rate, the expected mtDNA nucleotide diversity within a random-mating population or species is directly related to effective population size (Engels 198 1; Nei 1987). Perhaps the exceptional mtDNA restrictionsite polymorphism in menhaden relates to their enormous population size (probably numbering at least in the hundreds of millions at present). [Caution is required, however, because current census sizes of species are often poor guides to evolutionary effective sizes (Avise et al. 1988).]. On the other hand, chuckwallas are currently much less numerous. Perhaps their mtDNA diversity relates to extensive historical population subdivision, which has the effect of buffering mtDNA lineages against extinction and of increasing total sequence diversity within a species (Avise et al. 1984). In principle, other classes of potential explanations for the mtDNA hypervariability in either menhaden or chuckwallas might involve enhanced effective mutation rates. Although we cannot be certain of the evolutionary explanation(s) for the exceptionally high genetic diversity in these fishes and lizards, the variation does present an opportunity for recognition of individuals by mtDNA markers. The total number of distinct mtDNA genotypes in the menhaden (or chuckwalla) species must be enormous. For example, from our sample of menhaden, we estimate mean sequence divergence between individuals to be p = 0.024. Thus, a randomly drawn pair of menhaden are expected to differ at -400 mtDNA nucleotide positions. Taking 400 as an extremely conservative estimate of the number of variable nucleotide positions within the species, and assuming that these positions are free to vary (between even two of four nucleotide states) independently of one another, we find that the number of possible mtDNA sequence permutations is a2400, a number vastly larger than the total species census. While the assumption of independent variation among sites is very unlikely to hold for a presumably nonrecombining molecule such as mtDNA (unless parallel or convergent evolution of sites is rampant), nonetheless a great many genotypes clearly must be present within the species. Additional distinctions among individual fish derive from the mtDNA size differences. The capacity to distinguish individuals by mtDNA is a function of the discriminatory power of the assay, as well as a function of the inherent variability in the molecule. The level of polymorphism in menhaden and chuckwallas is exceptional but nonetheless merely falls at the high end of a continuum of mtDNA variabilities observed in other species (table 2). Thus, if sufficient effort were expended in mtDNA assays [e.g., through use of either a battery of four-base enzymes (e.g., see Cann et al. 1984) or direct nucleotide sequencing of large (or hypervariable) regions of the genome (e.g., see Wrischnik et al. 1987)], it should be possible to extend mtDNA fingerprinting capabilities to less variable organisms as well. In higher animals, mtDNA is transmitted predominantly through female lines (Gyllensten et al. 1985; Avise and Vrijenhoek 1987; but see Satta et al. 1988). mtDNA fingerprints arising under this unisexual, clonal mode of inheritance thus differ in several respects from DNA fingerprints based on recombining, bisexually transmitted

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Table 2 mtDNA Genotypic Diversities (arranged by rank order) Observed in Conventional Restriction-Site Surveys of Various Species GENOTYPIC DIVERSITY

No.OF

SPECIES (common name) Brevoortia tyrannus (menhaden fish) . . . . . Sauromalus obesus (chuckwalla lizard) . . Peromyscus maniculatus (deer mouse). . . . . Lepomis gulosus (warmouth sunfish) . . . . . . Peromyscus polionotus (old-field mouse) . . . Geomys pinetis (pocket gopher) . . . . . . . . . Agelaius phoeniceus (red-winged blackbird). Amia calva (bowfin fish) . . . . . . . . . . . . . Lepomis punctatus (spotted sunfish) . . . . . . Limulus polyphemus (horseshoe crab) . . . . . Lepomis microlophus (redear sunfish) . . . . . Opsanus tau (oyster toadfish) . . . . . . . . . . . . Anguilla rostrata (American eel) . . . . . . . . Arius.felis (hardhead catfish). . . . . . . . . . . . .

Individuals 33 51 135 74 68 87 127 75 79 99 77 43 109 60

Different mtDNA Genotypes

[n(l - cj-‘)/PI - l] (max. possible = 1.0)

32 35 61 32 22 23 34 13 17 10

0.998 0.983 0.974 0.962 0.889 0.867 0.810 0.806 0.788 0.707 0.675 0.578 0.538 0.473

5

23 11

NOTE.-All studies are from the Avise lab and involve roughly similar screenings of mtDNA by using primarily Sbase and 6-base cutting endonucleases. References can be found in Avise et al. (1987).

nuclear genes. First, nuclear DNA fingerprints (obtained, e.g., from Jeffreys et al’s or M 13 probes) are normally scored only as “same” or “different” between individuals. While particular suites of fragments in nuclear fingerprints can be traced across a single generation to address questions of parentage, further estimation of relatedness among individuals is seldom possible (Lynch 1988). In contrast, because mtDNA mutations accumulate along clonal lineages, the mtDNA fingerprints can be aligned with respect to a range of evolutionary distances (Avise et al. 1987) and hence can be used to estimate matriarchal relationships at many levels in an intraspecific pedigree. Second, nuclear DNA fingerprints obtained from the Jeffreys et al. or M 13 probes are normally scored off a single gel. In contrast, the mtDNA fingerprints typically require amalgamation of information from a number of gels and hence are more labor intensive to generate. However, the natural partitioning of “raw” mtDNA data into a number of subsets (different enzyme profiles) can prove highly beneficial in terms of scoring, interpreting, cataloging, and comparing fingerprints from a large number of individuals. Third, mtDNA is maternally inherited and cannot be used to assess paternity, one of the primary applications of nuclear DNA fingerprinting. Nonetheless, genetic assignment of maternity can also be of interest, particularly in oviparous species where embryonic development takes place outside the female’s body. mtDNA fingerprinting should find special application in situations where maternity is in question. A related class of applications might involve assessment of the degree of dispersal and gene flow in matrilineally related individuals and their progeny. In the future, it seems likely that additional molecular approaches will become available to generate individual-specific genetic markers and that fingerprinting methods may thus be tailored to suit the needs of a particular study. Nonetheless, mtDNA

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will remain of special interest for genetic fingerprinting because of the unusual diagnostic properties that stem from its maternal mode of inheritance. Acknowledgments

We thank Tom Jones, Randy Babb, Mac Rawson, Paul Christian, Bruce Comyns, and Tom Sminkey for help in collecting specimens. Bill Nelson provided excellent assistance with data acquisition, and Marty Ball with data analysis. Research was supported by an NIH training grant (to B.W.B.), by contract DE-AC09-76SROO-8 19 between the U.S. Department of Energy and the University of Georgia, and by NSF grants BSR-8603775 and BSR 8805360. LITERATURE

CITED

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October 19, 1988; revision received December 2, 1988