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A Comprehensive Study of Genic Variation in Natural Populations of Drosophila melanogaster. IV. Mitochondrial DNA Variation and the Role of

History ZIS. Selection in the Genetic Structure of Geographic Populations

Lawrence R. Hale’ and Rama S. Singh Department of Biology, McMaster University, Hamilton, Ontario L8S 4N2, Canada

Manuscript received October 30, 1990 Accepted for publication May 16, 1991

ABSTRACT Preliminary studies with restriction fragment length polymorphisms of mitochondrial DNA

(mtDNA) in natural populations of Drosophila melanogaster revealed considerable variation in terms of nucleotide sequence and overall size. In this report we present data from more isofemale lines and more restriction enzymes, and explore the utility of the data in inferring a colonization history of this species. Size variation in the noncoding A + T-rich region is particularly plentiful, with size variants occurring in all restriction site haplotypes in all populations. We report here classes of small-scale mobility polymorphisms (apparent range of 20 bp) in specific restriction fragments in the coding region. The variation in one such fragment appears to be generated even more rapidly than in the noncoding region. On the basis of the distribution of restriction site haplotypes, the species range can be divided into three major regions along longitudinal lines: Euro-African populations are the most diverse and are taken to be oldest; Far East populations have a complex distribution of haplotypes; Western Hemisphere populations are the least diverse and are interpreted to be the youngest. The history inferred from mtDNA alone is remarkably similar to one based on several nuclear markers. The mtDNA haplotype distribution is also very different from that of allozymes in these same populations. We interpret this as further evidence that natural selection is still the most parsimonious explanation for the parallel latitudinal allozyme clines in this species.

T HE pattern of genetic variation in natural populations of species is a result of a complex inter-

play of natural selection and the effects of species history. Geographical routes of colonization, gene flow, and variation of population size (as it pertains to random genetic drift) are examples of historical factors which can influence genetic differentiation of populations. Historical information, therefore, is cru- cial to a study of the action of natural selection on the genetic structure of a species.

An important difference between the effects of natural selection and history on genetic variation is that natural selection will exert its influence differ- ently on individual loci, while the influence of history should be more or less equivalent on all loci. This feature of historical effects allows the inference of species history in two ways: a recurrent pattern of polymorphism, divergence and fixation of a number of loci may indicate the influence of history, or we can follow the fate of a single neutral marker.

Mitochondrial DNA (mtDNA) is an important tool in the analyses of phylogenies of closely related species (DESALLE and GIDDINGS 1986; GEORGE and RYDER 1986), of gene flow between hybridizable sibling spe-

Island, Charlottetown, Prince Edward Island, CIA 4P3, Canada.

Genetics 129: 103-117 (September, 1991)

’ Present address: Department of Biology, University of Prince Edward

cies and subspecies, particularly with respect to geographic zones of contact (FERRIS et a2 1984; CARR et al 1986; HARRISON, RAND and WHEELER 1987; SPOL- SKY and UZZEL 1984), and population structures of individual species (SAUNDERS, KESSLER and AVISE 1985; LANSMAN et al 1983; LATORRE et al. 1988). mtDNA is appropriate because of its uniparental in- heritance, nonrecombinogenicity, small size, and ele- vated rate of mutation (reviewed in AVISE et al. 1987). A much more contentious issue is whether intraspecific sequence variants of mtDNA are selectively neutral. The evidence for this is largely conjectural. The extreme importance of the mitochondrial gene prod- ucts implies a necessary conservation of their genes. Empirical support for this notion has come from interspecies sequence studies (BROWN and SIMPSON 1982) and experiments on cyto-nuclear associations (CLARK 1985; CLARK and LYCKEGAARD 1988). It is likely that most “selectable” mutations are therefore deleterious.

In this paper, we use restriction analysis of mtDNA polymorphism to infer species history in Drosophila melanogaster. In a preliminary report (HALE and SINGH 1987), we demonstrated that worldwide populations of D. melanogaster show a much greater degree of population structure for mtDNA restriction haplotypes than for allozyme variants. Here, we ex-

104 L. R. Hale and R. S. Singh

TABLE 1

Isofemale stocks of D. melanogaster

Population No. of lines Place of origin Date Source

France (FRA) 12 Villeurbanne, France 1978 England (ENG) 8 Covent Gardens, United King- 1980

West Africa (WAF) Central Africa (CAF) India (IND)

Korea (KOR)

Japan UAP) Taiwan (TAI)

Vietnam (VIE) Australia (AUS)

British Columbia (BC)

Ottawa (OTT)

Hamilton (HAM)

Massachusetts (MAS)

Florida (FLO) Texas (TEX) California (CAL) Argentina (ARC)

11 5 9

11

10 8

6 6

6

12

5

6

7 11 6 5 -

144

dom Benin Brazzaville, Congo Varanasi, India

Seoul, Korea

Jume, Japan Taipei, Republic of China

Ho-Chi-Minh City, Vietnam Fairfield, Australia

Pt. Coquitlam, British Colum-

Ottawa, Ontario, Canada

Hamilton, Ontario, Canada

Amherst, Massachusetts

Miami, Florida Brownsville, Texas Lakeside, California LaPlata, Argentina

bia, Canada

1978 1978 1983

1979

1980 1977

1978 1980

1983

1978

1977

1978

1983 1978 1982 1980

J. R. DAVID, CNRS, France M. KIDWELL, University of

Arizona J. R. DAVID J. R. DAVID B. N. SINGH, Baranas

Hindu University, India R. PAIK, Seoul National

University M. KIDWELL F.-J. LIN, Taiwan National

University J. R. DAVID P. A. PARSONS, LaTrobe

University, Australia A. BECKENBACH, Simon

Fraser University G. CARMODY, Carleton

University R. A. MORTON, McMaster

University D. HICKEY, University of

Ottawa M. KIDWELL D. HICKEY M. KIDWELL S. COSCARON, LaPlata,

Argentina

tend that analysis by means of an increased sample size from the same populations, an increased number of enzymes, and resolution of the full restriction pattern in the case of four-cutters. While the overall conclusions of our earlier paper are confirmed, there are some differences. The primary finding is that we are able to identify patterns of mtDNA variation that are in agreement with the colonization history, by mtDNA alone.

MATERIALS AND METHODS

Stocks used: The D. melunoguster stocks used in this study are summarized in Table 1. A total of 144 lines from 18 geographic locations were used in this study. These isofemale lines have been maintained in the laboratory at 18". Some lines were as old as 9 years at the time of mtDNA analysis, but this should not matter as the principle concern of long-term maintenance of isofemale lines, the stochastic loss of alleles, does not apply to mtDNA. Our preliminary mtDNA study (HALE and SINCH 1987) was done on a subset of the same lines.

mtDNA isolation and restriction analysis: Purification of mtDNA was done by a protocol extensively modified from LANSMAN et ul. (1981). Samples of 1-2 g of live and frozen adult flies were homogenized by hand in a Pyrex tissue grinder in 15 ml of homogenization buffer (0.1 M Tris (7.5), 0.01 M EDTA (7.5), 8% (w/v) sucrose), and then in a motor driven Potter-Elvehjem tissue grinder (Wheaton)

with a loose-fitting Teflon pestle. Homogenate was spun for 5 min at 3,000 rpm in a Sorval SS-34 rotor to sediment unbroken nuclei and cellular debris. The supernatant was spun for 20 min at 12,000 rpm to yield a pellet enriched for cytoplasmic constituents, including mitochondria. The pellet was resuspended in 3.7 ml of STE buffer [0.1 M Tris (7.5), 0.01 M EDTA (7.5), 0.05 M NaCI]. Suspended mitochondria were lysed by adding 150 pl of 10% SDS solution (in STE). A 4.0-ml sample of lysate was collected, to which 4.2 g of dry CsCl and 200 pl of 200 mg/ml ethidium bromide solution were added. This was spun for 12-1 5 hr in a Beckman Vti65.2 rotor at 55,000 rpm at 17".

mtDNA was collected by side puncture of the tube. Ethid- ium bromide was removed by washing the sample with single volumes of NaC1-saturated isopropanol until the orange tint was gone. The mtDNA was then precipitated away from the CsCl by adding two volumes of water and five volumes of 95% EtOH, chilling at -20" for at least 2 hr, and centrifug- ing. Purified mtDNA was then resuspended in 50 PI of T E buffer [0.01 M Tris (7.5), 0.001 M EDTA (7.5)].

mtDNA was digested with a series of restriction enzymes, according to manufacturers' protocols. We used 10 enzymes of varying recognition sequence length: 4 bases (DdeI, HuelII, Hinfl, HpuII, MboI, TuqI); 4.5 bases (AvuII); 6 bases (EcoRI, HindIII, XbuI). Restriction fragment ends were filled in with Klenow (Pharmacia) and the appropriate ["PI dNTP. Unincorporated radioactive nucleotides were removed by increasing the sample volume to 100 PI with TE, and passing through a 1.0-ml mini-column of G25-150 Sephadex (Sigma). The sample was ethanol precipitated in

mtDNA Variation in D. melunoguster 105

1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 p B R

FIGURE 1 .-Ag;~rose gel ( 1 '36) of Mho1 digests of D. melanogaster rntDNA. Hindlll-cut lambda-DNA is the size standard; fragment sizes are denoted in the margin. Lanes 1 , 3, 10: Mbo-D; lanes 2, 7, 8: Mbo-A; lane 6: Mbo-K.

a micro-centrifuge tube, and resuspended in 10 rl of TE. Restriction fragments were separated by electrophoresis

on 0.6-1 .O% agarose (Sigma) (Figure 1) and 8.5% acrylamide (Bio-Rad) gels (MANIATIS, FRITSCH and SAMBROOK 1982) (Figure 2). The 5.0% acrylamide denaturing gels were made by adding urea (Bio-Rad) to the acrylamide solution to a final concentration of 8%. Gels were vacuum-dried onto 3 MM Whatman filter paper and autoradiographed for 1-2 days using X-AR or X-RP film (Kodak).

Data handling: The full restriction pattern for any enzyme was visualized by using agarose gels to resolve fragments to 0.5 kbp, and acrylamide gels to resolve fragments smaller than 0.5 kbp. For each enzyme that revealed restriction site polymorphism, each variant pattern was assigned an upper case letter designation (e.g., Avu-A, Avu-B, etc.). The number of variable and nonvariable sites were inferred by reconstructing the minimum mutational pathways between variants of each enzyme. Most restriction sites could be mapped using the published sequence for the coding region of D. melunoguster mtDNA (DEBRUIJN 1983; GARESSE 1988; 13,215 bp of approximately 14,200 bp). The Univer- sity of Wisconsin Genetics Computer Group analysis package (DEVEREUX, HAEBERLI and SMITHIES 1984) was used to identify restriction sites in the sequence. Potential restriction sites (i.e., those with a single incorrect base) were identified to allow mapping of observed sites that are not in the published sequence. Some sites could only be mapped to two or more possible positions.

Estimates of mtDNA sequence divergence were calculated for three categories: between-population and between- haplotype divergence, and within-population diversity. Pair- wise measures of between-haplotype diversity were calculated using the maximum likelihood estimator of NEI (1 987) (dv; equations 5.50, 5.3). Estimates of variance were calculated using equations 5.44 and 5.51 of NEI (1987). These formulas consider the number of shared/nonshared sites for sets of enzymes with particular lengths of recognition sequence.

Intrapopulational diversities for the 18 populations (d,) were estimated according to equation (1) of NEI (1982), which is simply an average of the haplotype distances of all possible pairwise comparisons of individuals in the population. Estimates of pairwise distances between populations (d,) is calculated in a similar fashion (equation 10.20 of NEI 1987), except that pairwise comparisons are made between individuals sampled from different populations.

525 404

248

160

90

FIGURE 2.-Native acrylamide gel (8.5%) of MboI digests of D. melanoguster mtDNA. Hpall-cut pBR322-DNA is the size standard; the sizes of a few of these fragments are denoted in the margin. Lane 6 (Mbo-K) is the only pattern in this gel to show restriction polymorphism at this level of resolution.

Construction of a Wagner maximum parsimony network of observed haplotypes was done using the MIX program of the PHYLIP package (v. 3.1), kindly provided by JOE FELSENSTEIN. Several runs of MIX were performed using different random number seeds.

RESULTS

Size variation: Many cases of electrophoretic mobility polymorphism of D. melanogaster mtDNA fragments that are not attributable to gain/loss of restriction sites were observed. Insertion/deletion of sequences of variable length have been documented for mtDNA of many different organisms (POWERS, PLATZER and HYMAN 1986; BERMINCHAM, LAMB and AVISE 1986; WALLIS 1987; WOLSTENHOLME and DAWID 1968). Polymorphism of this sort falls into two classes, both of which were observed here. The more frequent class is sequence insertion/deletion in the noncoding region that contains the start site for tran- scription. In Drosophila, this region is rich in A-T base pairs (95%; KLUKAS and DAWID 1976), and called the A + T-rich region; it is homologous to the D-loop region of non-Drosophilids. Variation in the coding region of mtDNA is is rarer because of its extremely compact organization.

We have already documented size variation in the noncoding A + T-rich region of D. melanogaster mtDNA (HALE and SINCH 1986), which yielded a total size range of 18.1 to 19.9 kbp. We found that: (1) each restriction haplotype occurring more than once harbors size variation in the A + T-rich region;


TABLE 2

Number of isofemale lines (homoplasmic/heteroplasmic) showing A+T-rich region size variation in different restriction haplotypes in D. melanogaster

Size class (kbp)

Haplotype# 18.2 18.6 18.7 18.8 18.9 19.0 19.1 19.2 19.3 19.4 19.5 19.6 19.9 TI

13 13

1 6

6 5

7 1

2916 12/2 1/3 1/2 2/2 212 0/1 1/0 110 110 59 8 1 IO 0/1 3/0 o/ 1 5

1 212 0/2 110 1/1 312 0/1 2/0 2 1/0 912 o/ 1 1/0 011 3 1 IO 4 01 1 5 111 210 01 1 1 /o

3/0 2/1

1 IO

9 o/ 1 o/ 1 110 2 10 1 / O 2/0 3 1 1 1 /O 1 12 3/2 1/1 l / O o/ 1 1 /O 8 13 o/ 1 3/1 4 14 1 /o 1 15 1 /O 1 16 1 17 1 / 1 o/ 1 3 18 1 IO 1 19 1 /o 1 20 1 /o 1 21 0 22 210 1 / 1 110 0/1 5 23 1 IO 1

skew in the distribution of size classes (Figure 3). The

feature in the frequency distribution that is of interest in light of the results of SOLIGNAC, MONNEROT and MOUNOLOU (1 986). The new distribution may be mul- timodal. There are peaks that are higher than those immediately surrounding at 18.6, 19.1, 19.5 and 19.9 kbp. In their study of mtDNA of other species of the

3 extended sample does, however, reveal an additional

L melunoguster group, SOLIGNAC, MONNEROT and - MOUNOLOU (1986) found that a tandemly repeating unit of 450 bp was responsible for a considerable amount of the size variation seen in those species. Successive additions of this repeat units to the 18.6-

n nnnn- l l 10s 10.7 10s 10.9 I9D 19.1 192 19s 19.4 19d 19s 19.9

mtDNA size class (kbp) FIGURE 3.-Frequency distribution of A + T-rich region size

variants in D. melanogaster. In calculating frequencies, the variants in heteroplasmic lines were given half the weight of those in homoplasmic lines.

(2) several isofemale lines were found to be stably heteroplasmic for mtDNA size variants, but not site variants (with one exception); and (3) there was a strong skew in the frequency distribution of mtDNA size classes toward the smaller sized molecules.

The extended sample of the current report yielded nothing to contradict the conclusions of the earlier paper. All nonunique haplotypes harbor size varia- tions, and many lines were heteroplasmic for size variants (Table 2), and there is a strong, significant

kbp size class would result in the sizes commensurate with the peaks in the distribution.

Smaller mobility polymorphisms were also observed in mtDNA fragments from the coding region. The most pronounced case is that of an MboJ fragment that normally appeared on the acrylamide gels at an apparent size of 0.26 kbp (Figure 4A). Within the resolving power of the gels, this fragment appeared in six separate positions, between 0.25 and 0.28 kbp.

Variation in the fragment was observed to be even more common than A + T-rich region variation, as haplotypes of a single A + T-rich size class showed some variation in this fragment. For example, all mtDNAs sampled from Japan were of haplotype #7 and 18.6 kbp in length. Yet these lines all showed mobility polymorphism of the 0.26-kbp MboI frag-

mtDNA Variation in D. melanogaster 107

A

B 1 2 3

5 6 P

0 20"

0 24

0 I8

4 0.41

(kbp)

,. 0.36 * - 0.35' - 0 33 ' 0.31

_I 0 27 ,-0.26'

FIGURE 4.-Native acrylamide gels (8.5%) showing examples of apparent size variation in small fragments. The sizes of all mtDNA fragments in these regions are denoted in the margin. The positions of the fragment showing small-scale variation are indicated in each lane by an arrow. (A) Variation in the 0.26-kbp Mbol fragment. Four size classes are apparent. (B) Variation in the 0.34-kbp Tu91 fragments. Three different fragments show variation.

ment (in fact, Figure 4A is taken from lines from Japan). It would seem therefore, that variation in this fragment is generated much more quickly than in the A + T-rich region. We noted no instances of lines heteroplasmic for variants in this fragment. The frequency distribution of variants (Figure 5 ) shows a skew toward the smaller sizes, but the peak (kurtosis) is clearly not as strong as strong as for the A + T-rich region variants.

Three TuqI fragments also demonstrated mobility polymorphisms on acrylamide gels (Figure 4B), similar in magnitude to that observed with the MboI fragment. But the variants from the common type were restricted to haplotype #l. As will be discussed later, haplotype #1 is likely the oldest extant mtDNA haplotype in this species. Therefore, the TuqI fragment

"1

x3 266 269 272 275 270 281 284

mtDNA size class (bp)

FIGURE 5,"Frequency distribution of apparent size variation in the 0.26-kbp MboI fragment. Size classes are based on the "apparent' size of the fragment within the level of resolution, and is not intended to suggest the precision of the measurements.

variants are likely generated more slowly than A + T- rich region variants.

MboI digests of D. melunoguster mtDNA were ran on denaturing acrylamide gels ( i e . , 8% urea) to deter- mine whether the mobility polymorphisms were due to insertion/deletion of nucleotides, or conformational changes resulting from certain types of base substitution (SINGH, NECKELMAN and WALLACE 1987). Unfortunately, the alltoradiographs for these gels were not of good quality and quite uninformative (not shown).

Observed haplotypes: Among the 144 lines studied, 23 composite restriction types (haplotypes) were identified (Table 3). Most of the differences between haplotypes were fairly straightforward; mutational relationships between restriction patterns for individual enzymes could be inferred without a restriction map. A total of 20 polymorphic sites were ultimately in- voked as the minimum number necessary to define all 23 haplotypes.

The published D. melanogaster sequence encompasses 13.2 kbp, all of which is outside the A + T-rich region. Of the 100 monomorphic restriction sites, 94 could be mapped within the sequenced portion. Since many of the polymorphic restriction sites had to be identified by searching within the sequence for locations that mismatched the enzyme recognition sequence by one base, only 12 of the 20 polymorphic restriction sites could be unambiguously assigned to a particular location within the sequenced portion. The other eight could only be mapped to alternative positions within or beyond the sequenced portion.

Phylogenetic analysis: The 20 X 23 matrix (20 variable sites, 23 haplotypes; Table 3) was used to find the most parsimonious network of the observed h a p lotypes. Several runs of the MIX program produced two networks that differ only by the position of h a p lotype #6 (Figure 6). An important feature of this tree is that there is evidence of considerable repeat change at several sites. The depicted network requires 30 mutational events among the 20 polymorphic restriction sites. Among those sites determined to have


TABLE 3

Presence (Wabsence (0) matrix for variable restriction sites in D. melanogmter restriction haplotypes

AuaIl HaeIII abcd Haplotype

TaqI Mbol abc

H i d ab abcde abcd

Ddel ab

AAAABB-1 AAAAFB-2 AAEAAC-3 AAEABB-4 AAEEBB-5 ABAAAB-6 ABBAAC-7 BBBAAC-8 CAADCA-9 CAADCB-I 0 EBAAAC-11 EBABCB-12 EBABDB- 13 EBAKCB-14 EBBBAC-15 AAAAAC-I 6 AAACBB-17 AAEEBC- 18 ABABAB-19 ADAABC-20 DCACBB-2 1 ABABBA-22 ABABBA-23

1111 1111 1111 1111 1111 1111 1111 0111 1100 1100 1101 1101 1101 1101 1101 1111 1111 1111 1111 1111 1011 1111 1111

100 10 100 10 100 00 100 00 100 00 000 10 000 11 000 11 100 10 100 10 000 10 000 10 000 10 000 10 000 11 100 10 100 10 100 00 000 10 101 10 110 10 100 11 000 10

01001 01001 01001 01001 01 101 01001 01001 01001 1101 1 1101 1 01001 11001 11001 11000 l l0Ol 01001 0000 1 01 101 11001 01001 00001 01001 11001

1100 1000 0100 1100 1100 0100 0100 0100 1110 1110 0100 1110 1111 1110 0100 0100 1100 1100 0100 1100 0100 0100 1100

10 10 11 10 10 10 11 11 00 10 11 10 10 10 11 11 10 11 10 11 11 11 00

FIGURE 6.-Wagner parsimony network for mtDNA restriction haplotypes of D. melanogaster giving detailed information on the particular restriction site changes. Haplotype #6 is in parentheses to denote that there are two equally likely positions, both of which are shown. The tree is

meaning. The inferred restriction site changes between haplotypes are denoted by a single upper case letter designated the enzyme (A = AvalI, D = Ddel, F = Hinff, H = HaeIlI, M = Mbol, T = Tugl), a lower case letter designating the specific site (as in Table 3). and an arrow solely to indicate which direction the site is gained.

21

8 20

unrooted and branch lengths have no

15 3 4 19 14

5 6

18

undergone repeated change, 2 events are ascribed to of a TaqI site, are common in Southeast Asia (India one site, and 3 events at two others. TEMPLETON and Vietnam). A branch of two haplotypes, defined (1983) states that parallel losses of a site are more likely than parallel gains of a site. The only way to assess the relative occurrence of parallel gains and losses is to assign a “root” to this unrooted network. Haplotype #1 was chosen as the root because of its internal position in the network, and its high frequency in geographic locations that have been sug- gested as the place of origin of the species. With this root in place, seven of the parallel changes are losses, two are gains, with one other site experiencing a loss and a subsequent gain.

by the loss of an MboI site, is unique to West Africa. Finally, a branch of five haplotypes, defined by the loss of an Am11 site and the gain of a Hinfl site, is found predominantly in France, and to a lesser extent in Korea.

Genetic distances: The distribution of haplotypes among populations is shown in Table 4. Of the 23 haplotypes observed, 16 were observed in one population only, and of these six were detected in more than one line. Of the seven nonunique haplotypes, two (#2 and #22) were restricted to a single geograph-

One branch of three haplotypes, defined by the loss ical region (which are defined below), two (#1 and


TABLE 4

Distribution of D. melanogaster restriction haplotypes by geographic population

Western Hemisphere Euro-African Far East Haplo- type OTT HAM MAS FLO BC TEX CAL ARG ENG FRA WAF CAF IND KOR JAP TAI VIE AUS n

1 1 3 3 5

1

2 3 1 4 5 1 6 1 7 1 1 5 6 7 3 9 6 8 5 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 1

n 1 2 5 6 7 6 1 1 6 5

1 3

4

1 2 1

3

1 1

2 1

8 12 11

1 6 8

9 5

10 1

1

1

2

5 9 11 10 8 6

13 14

1 9 6 1

60 5 2 3 1 8 4 1 2 1 3 1 1 1 1 5 1

144

# 15) were at high frequency in one region with only a single observation in another, and one (#9) was found in two regions in a single line each. Only two haplotypes (#7 and #12) have a wide distribution in that they are found in all three regions.

These distributions suggest considerable endemism of haplotypes. However, and alternate depiction of the phylogenetic network, which shows the localities in which each haplotype was found (Figure 7), suggests otherwise. There is only limited geographic clustering of haplotypes along monophyletic branches. This is in sharp contrast to the observation in our earlier paper (HALE and SINCH 1987), where we found clear evidence of geographic associations. We place much greater confidence in the present analysis since we were able to visualize virtually the complete set of fragments generated by the four-cutter enzymes.

The presence/absence matrix was also used to generate measures of genetic distance between haplotypes (dij). These values are shown in Table 5 together with the minimum number of site differences between each pair (these are not corrected for parallel gain/loss of sites as indicated by the phylogeny). The genetic differences range from 0.00108 for two haplotypes that differ by the presence/absence of a single four-cutter site, to 0.0122 for haplotypes at opposite ends of the phylogeny and which do not share restriction patterns for any of the enzymes. The average pairwise value is 0.00053.

Geographic distribution of haplotypes: On the basis of intra- and interpopulational diversity of the samples, D. melanogaster populations can be roughly divided into three geographical regions. One of these regions is the Western Hemisphere. The other two are both in the eastern hemisphere. One encompasses all populations in Europe and Africa, and is hereafter designated as Euro-African. The other included the populations found in India and points east, and is hereafter designated as Far East.

Euro-African populations of D. melanogaster are evidently the most diverse. An average of 4.5 variant haplotypes were found in each sample, compared to 2.0 in the Far East and 1.8 in the Western Hemi- sphere. With the exception of Central Africa, these populations have at least two high frequency variants ( ie . , observed in more than one line). This results in an average intrapopulational diversity measure (d,) for these populations of 0.00106 (Table 6); this is larger than for either of the other two regions ( P < 0.05 for both comparisons: Mann-Whitney U-test). Further, at least one of these high frequency variants in each population is unique to it ( i e . , [#15] in Eng- land, [#lo] in France, and [#17] in West Africa). This results in a total regional diversity measure (d,) of 0.00196. Therefore, although one of the shared haplotypes (#1) is found at high frequency in 3 of the 4 populations, the populations are strongly differen-


p;; 10 $7- 19 Bc.FR4w.Aus

FIGURE 7,”Alternate depiction of Wagner parsimony networks giving detailed information as to the occurrence of each D. melanogaster mtDNA haplotype in geographic populations. Haplotype #6 is in parentheses to denote that there are two equally likely positions, both of which are shown. A single hatch along branches indicates one restriction site mutational event (see Figure 6 for details on site changes); no hatch indicates a branch of zero length.

tiated, with G,, ((d, - d,)/d,) of 0.459. The average interpopulational nucleotide diversity (dxJ is 0.00043.

The samples from Far East populations are individually less diverse than those from Euro-African populations. Most have one dominant haplotype. This results in a d, measure of 0.00056. However, the dominant haplotype differs among populations, so that over the region many different haplotypes reach high frequency. Six haplotypes were observed in more than one line sampled from this region, and of these four were restricted to one population. The d, value for the region is 0.00203, which is close to that seen in the Euro-African region. As a result, the G,, value for this region is 0.724, which is noticeably higher than that calculated for the Euro-African region. On this basis, Far East populations would seem to be more differentiated, although individually less diverse. However, the average distance (dJ is not significantly greater than that seen in Euro-African populations ( P > 0.05).

The Western hemisphere populations are the least diverse of all the regions. Samples have either a single haplotype, or a dominant one with 1-3 singletons. As a result, the regional d, value (0.00033) is quite low in comparison to that of the Old World (0.00076). The dominant haplotype is the same in 7 of 8 populations (#7) and so the d, value (0.00059) for the region is quite low as well. This also contrasts sharply to the situation for either Euro-African or Far East populations ( i e . , Old World), which is a result of many different haplotypes being found at high frequency.

The low degree of interpopulational differentiation of Western Hemisphere populations is best shown by the Nei genetic distance (d,) between populations.

The average distance between Western Hemisphere populations is 0.001 18, which is substantially smaller than corresponding values from the Euro-African and Far East regions (0.00434 and 0.00462, respectively).

The fixation of haplotype #8, which is one mutational step removed from #7, in Argentina shows that some differentiation is starting to occur. Still, the rest of the variation in this region is due to haplotypes that are not derived from haplotype #7.

DISCUSSION

mtDNA variation and the colonization history of D. melanogaster: A proposed colonization history of D. melanogaster, based on the cumulative data from studies of chromosomal arrangements, allozymes, morphometric and physiological traits, has recently been proposed by DAVID and CAPY (1988). They identified three categories of populations, ranked by age, which correspond to three major episodes in the expansion of this species. The oldest of these, the “ancestral” populations, are those found in tropical Africa and were so designated largely because allozyme diversity is greater (SINCH and RHOMBERG 1987), and other melanogaster subgroup species are endemic to this region.

The second category, that of “ancient” populations are those which presumably were colonized by the first waves of migration from Africa, notably Europe and Asia. They are believed to be younger than the ancestral ones, as evidenced by their generally lower degree of intrapopulational variability. The success of D. melanogaster in both temperate and tropical cli- mates is taken as evidence of natural selection acting over a long period of time. The finding of a Far East “race” of D. melanogaster, which differs from Euro- pean and African stocks in several morphological and physiological traits, is taken to indicate a long separa- tion of the two regions. The range of the Far East race includes Japan, Taiwan, Vietnam and Sri Lanka, but not Australia. Far East populations are not quite as polymorphic as their counterparts in Europe and Africa for several other traits (e .g . , allozymes, chromosomal arrangements).

The Australian population was placed in the third category, that of “new” populations. New populations are defined as those introduced by humans, presumably within the last 200-300 years, and include Aus- tralian and North American populations. DAVID and CAPY (1988) suggest that the colonization of North America by D. melanogaster was a two-step process: an initial African introduction was followed by the introduction of cold-adapted European flies.

Using mtDNA, the D. melanogaster species range can be differentiated into distinct regions, separated along longitudinal lines. (Due to the virtual ubiquity of size variants across haplotypes and populations,

I. 2 8 ? 1 &I 4 0

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TABLE 7 0.00 I d t A summary of variation statistics for mtDNA and allozymes

from D. melanogaster

ReKion Data type

Euro-African mtDNA Allozymes

Western Hemi- mtDNA sphere Allozymes

Far East mtDNA Allozymes

Worldwide mtDNA Allozvmes

intrapopulational Average

diversity (d,fH,)

0.106* 0.130

0.033* 0.101

0.056* 0.098

0.064* 0.102

Regional Fixation diversity index

(dtfHJ ( G s O

0.196* 0.459 0.132 0.106

0.059* 0.441 0.127 0.079

0.203* 0.724 0.1 12 0.097

0.188* 0.660 0.138 0.126

Allozyme data from SINGH and RHOMBERG (1987) and unpub- lished. * = XlO-'.

Allozyme genetic distance

FIGURE S.-Correlation of interpopulational genetic distance from mtDNA and allozyme analysis for nine D. melanogaster populations. Open circles denote comparisons within the Western Hem- isphere, which have been omitted from the calculation of regression and correlation (see text). Y = 0.0579 - (0.21)X. Correlation coefficient (r') = 1.4% ( P > 0.05).

almost all of the phylogeographic inferences made by mtDNA is based on site variation. Those instances where size variation is of some utility are noted in the following discussion). The three regions (Euro-Africa, Far East, and Western Hemisphere) are distinguished on the basis of levels of intrapopulational variability and interpopulational divergence. Euro-African populations are highly polymorphic and moderately divergent, Far East populations are much less polymorphic but strongly divergent, and Western Hemisphere populations are nearly monomorphic and very similar to each other. From this, an approximate colonization history of D. melanogaster can be derived.

Two lines of evidence suggest that Euro-African populations have a longer history than those of other regions. First, the region is the most diverse, due largely to unique haplotypes occurring at high frequency in three of the four populations studied. Sec- ond, haplotype #1 is at high frequency. This particular

I - 0 I

0 3 6 9 12

Geographic distance (km x 1000)

FIGURE 9.-Correlation of interpopulational mtDNA genetic distance and geographic distance between nine D. melanogaster populations. Open circles denote comparisons within the Western Hem- isphere, which have been omitted from the calculation of regression and correlation (see text). Y = 0.0689 - (2.7 X 10-6)X. Correlation coefficient (r') = 10.4% (P > 0.05)

haplotype is probably the oldest extant haplotype of D. melanogaster, as evidenced from its central position in the phylogenetic network (Figure 6), and the presence of coding-region size variation in a Tag1 fragment, which is lacking in all other haplotypes (Figure 4B). However, unlike gene-enzyme analysis which shows more unique alleles in the African populations, the mtDNA analysis does not distinguish between the European or African populations as being older than the other.

Of the three geographic regions, the history of the Far East is the least straightforward. The high d, value for the region suggests that, like the Euro-African region, the Far East populations are rather old. Yet the small d, indicates that, unlike the Euro-African populations, the Far East populations have not simply maintained stable numbers and accumulated new variants. There are, perhaps, two explanations for the observed population structure here. In the first possibility, all of the Far East populations could be derived from a single introduction. After a period of time to allow expansion and divergence throughout the region, individual stochastic events such as bottlenecks could produce the generally small d, and consequent high GSt. The second possibility stipulates several introductions to certain localities only. Thus, for in- stance, the three haplotypes found in India and Viet- nam constitute a monophyletic branch off haplotype #l.

The two unique Australian haplotypes (#11, #19) derive from the two highest frequency haplotypes, neither of which are specific to the Far East, at distant points along the phylogeny. This suggests that the Australian population has also a Euro-African origin and possibly has received immigrants from the West- ern Hemisphere, but has not yet differentiated to the


same degree as other Far East populations. The mtDNA complement of the sample from Japan

is particularly curious. First, the only restriction haplotype observed (#7) is one that is predominantly a “Western Hemisphere” type. While this immediately suggests that this population may have been colonized from North America, a Euro-African origin in this case can not be ruled out since #7 was observed in one Euro-African population. Second, size variation was restricted to the small-scale polymorphism in the MboI fragment (Figure 4A); no A + T-rich region size variation was detected, unlike all other populations. These results imply a relatively recent origin.

The observation of one strongly predominating haplotype (#7) in the Western Hemisphere indicates a low effective population size or directional selection leading to the fixation of one predominant type. It is likely that Western Hemisphere populations derive from a single introduction of flies that was monomorphic, or nearly so, for mtDNA in the recent past. There is, however, some diversity of mtDNA within the region. First, the Argentina population is fixed for a haplotype (#8) that is one mutational step removed from #7. Because the two haplotypes are close phylogenetically, it is reasonable to hypothesize that #8 was derived from #7 in Argentina.

The other haplotypes observed in the Western Hemisphere are all singletons which do not show close phylogenetic linkage to #7. Many of them are observed at high frequency in populations of other regions and we propose them to be spurious migrants from other localities, and not an established part of the normal mtDNA complement of their respective Western Hemisphere populations. All Western Hem- isphere populations have several size variants among, arguing that the populations have been established long enough to generate such variation. Yet samples of four of the seven populations revealed only haplotype #7. It would seem, therefore, that recurrent migration has not altered significantly the mtDNA composition of populations.

The histories proposed by the cumulative data and mtDNA alone are reasonably concordant, in that they both reveal three major episodes in the colonization by D. melanogaster. There are some differences in detail, most notably the inability of the mtDNA history to distinguish between European and African populations, and the observation of relationships among certain Far East populations by mtDNA alone.

An inconsistency lies with our finding of the Japa- nese population sample being monomorphic for haplotype #7, which is so prominent in the Western Hemisphere populations. This suggests that Japan has been recently colonized, although Japanese flies have the morphological characteristics of the Far East ‘race’. Intense selection for the Far East characteristics

is not unlikely as latitudinal variation in morphometric traits is thought to have occurred rapidly in Japanese populations of D. melanogaster (WATADA, OHBA and TOBARI 1986). Another possibility is that the Japanese archipelago could be subdivided for D. melanogaster from old and new introductions; our sample therefore would represent only the newer stock.

Second, DAVID and CAPY (1 988) suggest a two-step introduction of D. melanogaster into the Western Hemisphere, while the mtDNA indicates only one. The similarity of Northern temperate (Ottawa, Ham- ilton, Massachusetts) and Southern tropical populations (Florida, Texas, California) suggests that both derive from the same original introduction(s) of flies into the Western Hemisphere. Haplotype #7 is appar- ently a rare variant in Africa, and therefore it is somewhat surprising that, barring strong selection, it has been effectively fixed in the Western Hemisphere. Unless there are isolated pockets of flies with a high frequency of #7 in both Europe and Africa, it would seem extremely unlikely that two independent colo- nizing introductions from Europe and Africa into the Western Hemisphere would lead exclusively to #7.

EANES et al. (1 989) have also argued against a two- stage colonization of North America on the basis of restriction analysis of the GGPD locus from flies from temperate North America (New York State) and Eu- rope (France). They found that haplotypes character- ized by a 4.2-kbp insertion, which are at high frequency in Europe, did not appear in the temperate North American sample, and suggest that this is in- consistent with large-scale introduction of European genes into North America.

Comparison of mtDNA and allozyme based genetic structure: Many groups have undertaken sur- veys of allozyme variation in natural populations of D. melanogaster (ANDERSON and OAKESHOTT 198 1 ; BER- GER 1970; CAVENER and CLEGG 1981; FRANKLIN 198 1 ; OAKFSHOTT et al. 198 1 ; SINGH and COULTHART 1982; SINGH, HICKEY and DAVID 1982; VOELKER, MUKAI andJoHNSoN 1977). The most comprehensive is that of SINGH and RHOMBERG (1 987) in which 1 17 loci were surveyed to yield precise estimates of heterozygosity and geographic differentiation. In D. melanogaster, 15 populations were sampled, of which nine are the same as used in this mtDNA survey. Two main issues are raised in comparing the two systems; the overall geographic differentiation and structure of populations, and the comparative abundance and pattern of distribution of variants at individual loci.

Table 7 gives a summary of relevant measures with respect to population structure of allozymes and mtDNA. Two points stand out. First, there is compar- atively little difference between regions for the means of intrapopulational heterozygosity over loci. Popula- tions which are monomorphic for mtDNA (such as


Hamilton and Taiwan) are as heterozygous for allozymes as populations (such as France and West Africa) which have highly polymorphic mtDNA comple- ments. Second there is little difference in regional Ht or GSt. The G,, in Far East populations is not higher than in other regions, and the regional Ht is not lowest in the Western Hemisphere.

These comparisons indicate that the interpopula- tion differentiation of mtDNA is largely independent of allozyme variation. This is also seen in Figure 8, which is a plot of mtDNA distance (d,) vs. allozyme distance for population pairs. Initially, there is a positive correlation between these measures ( P < 0.05). However, all of the comparisons of populations within the Western Hemisphere, which are hypothesized to have shared a recent common history, cluster together near the origin. If these comparisons are omitted, then a negative correlation is the result ( P > 0.05).

Sixty-one of the 117 gene-enzyme loci in D. melanoguster were found to be polymorphic. The abundance and distribution of variation at polymorphic loci differs considerably from locus to locus. The distribution of values for interpopulational differentiation of loci (FSt) is strongly skewed. Although the range of values is from 0 to 0.68, there is a prominent mode within a bell-shaped distribution around F,, = 0.08, and then a long tail of higher values (SINGH and RHOMBERC 1987). Within the mode are loci with little total heterozygosity and ones with high heterozygosity. As such, differentiation is not a simple function of variability. In fact, loci with intermediate values of total heterozygosity are somewhat more differentiated between populations than those with a higher H , (SINGH and RHOMBERG 1987).

Among those allozyme loci with a higher F,, are 18 loci that show parallel clinal variation along temperate/tropical transects in each of the three regions (North America: OTT-TEX; Euro-African: FRA- WAF; Far East: TAI-VIE). SINGH and RHOMBERG (1987) showed that, although there is a positive correlation between allozyme distance vs. great circle geographic distance, it is weak because temperate populations are quite similar in their allozyme frequencies, while tropical populations differentiation is correlated with geographic distance. While the latitudinal clines in different continents have been ex- plained as being due to selection (SINGH and RHO" BERG 1987), the question nevertheless arises whether they could be due to large scale gene flow between continents and between regions within continents. The results for mtDNA are expected to shed light on this issue. First, there is no detectable correlation between mtDNA distance and geographic distance (Figure 9) (like Figure 8, there is a nonsignificant negative correlation if the population pairs within the Western Hemisphere are omitted). Second, the three

temperate populations (OTT-FRA-TAI) are com- pletely different from one another, as are the three tropical populations (TEX-WAF-VIE); there are no shared haplotypes among each set. Third, the pairs of populations from each region show sharply contrast- ing degrees of differentiation, concomitant with the general results for each region. The Western Hemi- sphere pair is almost identical, the Euro-African pair is moderately diverged, and the Far East pair is alter- nately fixed. As the temperate and tropical populations all appear to have different histories, the latitudinal allozyme clines observed in each continent can- not be historical in nature. Natural selection, in response to some latitudinally changing environmen- tal variable, remains the best explanation.

An important facet of the population ecology of D. melanogaster is its commensal relationship with humans. It then might be expected that the geographic distribution of variation of could show some concord- ance. DAVID and CAPY (1988) show that the geneti- cally inferred species history of D. melanogaster does largely coincide with important events in human history. Data on mtDNA and nuclear gene-enzyme variation in humans is normally presented in the context of racial groups, rather than simple geographical as- sociation. Allozyme variation shows that human groups are not strongly differentiated, and that Afri- can populations show the most discordance from other populations (LEWONTIN 1982). This is similar to what is seen in D. melanogaster (SINGH and RHOMBERG 1987). mtDNA in humans also seems to suggest an African orgin (CANN, STONEKING and WILSON 1987), as they show the greatest intra- and interpopulational diversity. Variation in mtDNA from Asian humans is also similar to that among D. melanogaster (Far East) in that the divergence within the region is not quite as high as for the African sample. Samples of mtDNA from nonaboriginal Western humans have typically been grouped with European samples (JOHNSON et al. 1983; CANN, STONEKINC and WILSON 1987), so comparisons with Western Hemisphere D. melanogaster are less meaningful.

Other studies comparing allozyme and mtDNA geographic variation (e.g. , SAUNDERS, KE~SLER and AVISE 1985; BICKHAM et a1 1989; OVENDEN and WHITE 1990; REEB and AVISE 1990) together with the present one, demonstrate that the distribution of variation can be quite different in mtDNA and allozymes. Given the possible role of selection in shaping allozyme variation profiles, mtDNA offers a useful means of deter- mining the historical factors affecting the genetic structure of species.

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to R.S.S., and an Ontario Graduate Scholarship to L.R.H.


LITERATURE CITED

ANDERSON, P. R., and J. G. OAKESHOTT, 1981 Geographic clines and climatic associations of Adh and a-Gpdh gene frequencies in Drosophila melanogaster, pp. 237-250 in Genetic Studies of Drosophila Populations, edited by J. B. GIBSON and J. G. OAK- ESHOTT. Australian National University, Canberra.

AVISE, J. C., J. ARNOLD, R. M. BALL, E. BERMINGHAM, T. LAMB, J. E. NEIGEL, C. A. REEB and N. C. SAUNDERS, 1987 Intraspecific phylogeography: The mtDNA bridge between population genetics and systematics. Annu. Rev. Ecol. Syst. 18:

BERGER, E. M., 1970 A comparison of gene-enzyme variation between Drosophila melanogaster and D. simulans. Genetics 6 6

BERMINGHAM, E., T. LAMB and J. C. AVISE, 1986 Size polymorphism and heteroplasmy in the mtDNA of lower vertebrates. J. Hered. 77: 249-252.

BICKHAM, J. W., S. W. CARR, B. G. HANKS, D. W. BURTON and B. J. GALLAWAY, 1989 Genetic analysis of population variation in the Arctic Cisco (Coregonus autumnalis) using electrophoretic, flow cytometric, and mitochondrial DNA restriction analyses. Biol. Pap. Univ. Alsk. 24: 112-124.

BROWN, G. G., and M. V. SIMPSON, 1982 Novel features of animal mtDNA evolution as shown by sequences of two rat cytochrome oxidase subunit I1 genes. Proc. Natl. Acad. Sci. USA 7 9 3246- 3250.

CANN, R. L., M. STONEKING and A. C. WILSON, 1987 Mitochondrial DNA and human evolution. Nature 325: 31- 36.

CARR, S. M . , S. W. BALLINGER, J. N. DERR, L. H. BLANKENSHIP and J. W. BICKHAM, 1986 Mitochondrial DNA analysis of hybridization between sympatric white-tailed and mule deer in west Texas. Proc. Natl. Acad. Sci. USA 83: 9576-9580.

CAVENER, D. R., and M. T . CLEGG, 1981 Temporal stability of allozyme frequencies in a natural population of Drosophila melanogaster. Genetics 9 8 61 3-623.

CLARK, A. G., 1985 Natural selection with nuclear and cytoplasmic transmission. 11. Tests with Drosophila from diverse populations. Genetics 111: 97-1 12.

CLARK, A. G., and E. M. S. LYCKEGAARD, 1988 Natural selection with nuclear and cytoplasmic transmission. 111. Joint analysis of segregation and mtDNA in Drosophila melanogaster. Genetics

DAVID, J . R., and P. CAPY, 1988 Genetic variation of Drosophila melanogaster natural populations. Trends Genet. 4: 106-1 11.

DEBRUIJN, M. H. L., 1983 Drosophila melanogaster mitochondrial DNA; a novel organization and genetic code. Nature 304: 234- 241.

DESALLE, R., and L. V. GIDDINGS, 1986 Disocordance of nuclear and mitochondrial DNA phylogenies in Hawaiian Drosophila. Proc. Natl. Acad. Sci. USA 8 3 6902-6906.

DEVEREUX, J., P. HAEBERLI and 0. SMITHIES, 1984 A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-397.

FANES, W. F., J. W. AJIOKA, J. HEY and C. WESLEY, 1989 Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster. Mol. Biol. Evol. 6 384-397.

FERRIS, S. D., R. D. SAGE, E. M. PRAGER, U. RITTE and A. C. WILSON, 1984 Mitochondrial DNA evolution in mice. Ge- netics 105 681-721.

FRANKLIN, I . R., 1981 An analysis of temporal variation at isozyme loci in Drosophila melanogaster, pp. 217-236 in Genetic Studies of Drosophila Populations, edited by J. B. GIBSON and J. G. OAKESHOTT. Australian National University, Canberra.

GARESSE, R., 1988 Drosophila melanogaster mitochondrial DNA:

489-522.

677-683.

118: 471-482.

gene organization and evolutionary considerations. Genetics

GEORGE, M., JR., and 0. A. RYDER, 1986 Mitochondrial DNA evolution in the genus Equus. Mol. Biol. Evol. 3 535-546.

HALE, L. R., and R. S. SINGH, 1986 Extensive variation and heteroplasmy in size of mitochondrial DNA among geographic populations of Drosophila melanogaster. Proc. Natl. Acad. Sci.

HALE, L. R., and R. S. SINGH, 1987 Mitochondrial DNA variation and genetic structure in populations of Drosophila melanogaster. Mol. Biol. Evol. 4 622-637.

HARRISON, R. G., D. M. RAND and W. C. WHEELER, 1987 Mitochondrial DNA size variation in Field Crickets across a narrow hybrid zone. Mol. Biol. Evol. 4: 144-1 58.

JOHNSON, M. J., D. C. WALLACE, S. D. FERRIS, M. C. RATTAZZI and L. L. CAVALLI-SFOZA, 1983 Radiation of human mitochondrial DNA types analyzed by restriction endonuclease cleavage patterns. J. Mol. Evol. 19: 255-271.

KLUKAS, C. K., and I. DAWID, 1976 Characterization and mapping of mitochondrial ribosomal RNA and mitochondrial DNA in Drosophila melanogaster. Cell 9: 6 15-625.

LANSMAN, R. A., R. 0. SHADE, J. F. SHAPIRA and J . C. AVISE, 198 1 The use of restriction endonucleases to measure mitochondrial sequence relatedness in natural populations. 111. Techniques and potential applications. J. Mol. Evol. 17: 214- 226.

LANSMAN, R. A., J. C. AVISE, C. F. AQUADRO, J. F. SHAPIRA and S. W. DANIEL, 1983 Extensive genetic variation in mitochondrial DNA's among geographic populations of the deer mouse, Peromyscus maniculatus. Evolution 37: 1-16.

LATORRE, A., E. BARRIO, A. MOYA and F. J. AYALA, 1988 Mitochondrial DNA evolution in the Drosophila obscura group. Mol. Biol. Evol. 5: 7 17-728.

LEWONTIN, R. C., 1982 ' Human Diversity. W. H. Freeman, New York.

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

NEI, M., 1982 Evolution of human races at the gene level, pp. in Human Genetics, Part A: The Unfolding Genome, edited by B. BONNE-TAMIR, P. COHEN and R. N. GOODMAN. Alan R. Liss, New York.

NEI, M,, 1987 Molecular Evolutionary Genetics. Columbia Univer- sity Press, New York.

OAKESHOTT, J. G., G. K. CHAMBERS, J. B. GIBSON and D. A. WILLCOCKS, 198 1 Latitudinal relationships of esterase 6 and phosphoglucomutase gene frequencies in Drosophila melanogaster. Heredity 47: 385-396.

OVENDEN, J. R., and R. W. G. WHITE, 1990 Mitochondrial and allozyme genetics of incipient speciation in a landlocked POPU- lation of Galaxias truttaceus (Pices:Galaxiidae). Genetics 124 701-716.

POWERS, T. O., E. G. PLATZER and B. C. HYMAN, 1986 Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite, Romanomermis culicivorax. Curr. Genet. 11: 71-77.

REEB, C. A., and J. C. AVISE, 1990 A genetic discontinuity in a continuously distributed species: mitochondrial DNA in the American oyster, Crassostrea virginica. Genetics 124: 397-406.

SAUNDERS, N. C., L. G . KESSLER and J. C. AVISE, 1985 Genetic variation and geographic differentiation in mitochondrial DNA of the horseshoe crab, Limulus polyphemus. Genetics 112: 61 3- 627.

SINGH, G., N. NECKELMANN and D. C. WALLACE, 1987 Conformational mutations in human mitochondrial DNA. Na- ture 329: 270-272.

SINGH, R. S., and M. B. COULTHART, 1982 Genic variation in

118 637-648.

USA 83: 88 13-88 17.

mtDNA Variation in D. melunoguster 117

abundant proteins of Drosophila melanogaster and D. pseudoob- scura. Genetics 102: 437-453.

SINGH, R. S., D. A. HICKEY and J. R. DAVID, 1982 Genetic differentiation between geographically distant populations of Drosophila melanogaster. Genetics 101: 235-256.

SINGH, R. S., and L. R. RHOMBERG, 1987 A comprehensive study of genic variation in natural populations of Drosophila melanogaster. 11. Estimates of heterozygosity and patterns of geographic differentiation. Genetics 117: 255-27 1 .

SOLIGNAC, M., M. MONNEROT and J.-C. MOUNOLOU, 1986 Con- certed evolution of sequence repeats in Drosophila mitochondrial DNA. J. Mol. Evol. 2 4 53-60.

SPOLSKY, C., and T. UZZELL, 1984 Natural interspecies transfer of mitochondrial DNA in amphibians. Proc. Natl. Acad. Sci.

TEMPLETON, A,, 1983 Phylogenetic inference from restriction en- USA 81: 5802-5805.

donuclease cleavage site maps with particular reference to the evolution of man and the apes. Evolution 37: 221-244.

VOELKER, R. A,, T. MUKAI and F. M. JOHNSON, 1977 Genetic variation in populations of Drosophila melanogaster from the western United States. Genetica 47: 143-148.

WALLIS, G. P. 1987 Mitochondrial DNA insertion polymorphism and germ line heteroplasmy in the Triturus cristatus complex. Heredity 58: 229-238.

WATADA, M., S. OHBA and Y. N. TOBARI, 1986 Genetic differentiation in Japanese populations of Drosophila simulans and D. melanogaster. 11. Morphological variation. Jpn. J. Genet. 61:

WOLSTENHOLME, D. R. and I. DAWID, 1968 A size difference between mitochondrial DNA molecules of urodele and anuran amphibians. J. Cell Biol. 39: 222-228.

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