The phylogeny of the Drosophila obscuru group of species has been the subject .... pseudoobscura and the two strains of D. algonquin have distinctive ...
Phylogenetic Reconstruction of the Drosophila obscura Group, on the Basis of Mitochondrial DNA’ Eladio Barrio, * Amparo Latorre, * >TAndrh Maya, * 9-fand Francisco J. Aya1a-f *Department de Gen&ica, Facultat de Biologia, Universitat de Valencia; and TDepartment of Ecology and Evolutionary Biology, University of California, Irvine
We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (afinis and obscura) does not correspond to the phylogeny of the group, which shows two well-defined clusters (the Nearctic afinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency
to evolve toward high A+T values. This leads to a “saturation” effect that ( 1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDNA restriction analysis has for the phylogenetic reconstruction of Drosophila species that are not closely related.
Introduction The phylogeny of the Drosophila obscuru group of species has been the subject of numerous investigations. Morphological considerations ( Buzzati-Traverso and Scossiroli 1955) as well as allozyme data (Lakovaara et al. 1972; Marinkovic et al. 1978; Cabrera et al. 1983; Loukas et al. 1984) support the existence of two subgroups: the afinis subgroup, which is exclusively Nearctic, and the obscuru subgroup, with both Nearctic and Palearctic species. However, the evidence for a single subdivision into two subgroups is weak, that is, the average genetic distance based on allozyme data is about the same between the uffinis subgroup and the Palearctic obscuru as it is between the Nearctic and the Palearctic obscuru (Lakovaara et al. 1976; Lakovaara and Keranen 1980). Both restriction analysis of mitochondrial DNA (mtDNA) (Latorre et al. 1988 ) and DNA-DNA hybridization of single-copy nuclear DNA (Goddard et al. 1990) instead support the existence of three subgroups: the Palearctic obscuru (which will be hereafter referred to as the “obscuru subgroup”), the Nearctic obscuru (“pseudoobscuru subgroup”) and the Nearctic c&nis (“afinis subgroup”). This triple subdivision is supported by biogeographical considerations that favor the obscuru colonization of the Nearctic region through the Bering Straits (Throckmorton 1975), as well as by some phylogenetic reconstructions based on allozyme data (Lakovaara and Saura 1982). A number of African species have recently been described (Tsacas et al. 1985) that, as supported by allozyme data (Cariou et al. 1988), make up a fourth subgroup, the microlubis. The mtDNA has been useful in the phylogenetic reconstruction of species that are closely related (for a review of the characteristics of mtDNA that make this molecule 1. Key words: evolution, ratesof mtDNA evolution, molecular clock, A+T saturation. Address for comzspondence and reprints: Francisco J. Ayala, Department of Ecology and Evolutionary Biology, University of California, Irvine, California 927 17. Mol. Bid.
Ed. 9(4):62 l-635. 1992. 0 1992 by The University of Chicago. All rights merved.
0737-4038/92/0904-0005.$02.00
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I
D.affinis
II
D.algonquin
LI
16000 bp
Ill
D.algonquin
HF
16000 bp
16000 bp
Mvnl Hae III Hpall Eco RI Hind Ill Hpal Pvu II Sacl %:l Xhol
Mvnl Hae Ill Hpall EcoRI Hind Ill Hpal Pvu II Sacl Sea I Xbal Xhol
Mvnl Hae Ill Hpall Eco RI Hind Ill Hpal Pvu II Sacl %Y Xhol
FIG. 1.-mtDNA restriction maps of the 16 haplotypes found in species of the Drosophila obscuru group. The circular genomes have been linearized at a MM-ThuI restriction site common to all haplotypes. The symbols on the left refer to the 13 enzymes used; only those enzymes that yield restriction sites are shown for each particular species. The fragment sizes are given in base pairs (bp); some are estimated by reference to homologous sites in the mtDNA sequences of D. yukuba (Clary and Wolstenholme 1985) and D. melunoguster (De Bruijn 1983; Garesse 1988).
excellent for this purpose, e.g., see Harrison 1989), such as species groups of Drosophila. Restriction-fragment comparisons have been used for studying the pseudoobscura subgroup (Hale and Beckenbach 1985) and the obscura group (Latorre et al. 1988); . restriction-map analysis for studying the melanogaster subgroup (Solignac et al. 1986), the obscura group (Gonzalez et al. 1990)) and Hawaiian species ( DeSalle and Giddings 1986); and DNA sequencing for studying the melanogaster siblings (Satta et al. 1987) and Hawaiian species (DeSalle et al. 1987). One outcome of these investigations ‘is evidence showing that the advantages of mtDNA for phylogenetic reconstruction decrease as the phylogenetic divergence of the species increases, so that biases appear when fairly divergent taxa are compared.
mtDNA Phylogeny of Drosophila obscura
IV
623
16000 bp
D. arteca
Mvnl Hae III Hpall Eco RI Hind Ill Hpal Pvu II Sacl %l Xhol
V
D. narragansett
16000 bp
VI
D. tolteca
15800 bp
Mvnl Hae Ill Hpall EcoRl Hind Ill Hpal Pvu II Sacl Sea I Xbal Xhol
Mvnl Hae Ill Hpall Eco RI Hind Ill Hpal Pvull Sacl Sea I Xbal Xhol
FIG. 1 (Continued)
We present here the results of a restriction-map analysis of the mtDNA of 13 species of the D. obscuru group, an analysis that extends our previous mtDNA study of the obscura group (Latorre et al. 1988 ) . We analyze 2 1 strains from 14 different taxa that include the @inis subgroup (five species), the obscura subgroup (five species), and the pseudoobscuru subgroup (three species, one with two subspecies). We use methods for phylogenetic inference that include bootstrap (Felsenstein 1985, 1990). and Methods Drosophila Species
Material
We analyze 21 different strains from 13 species and one subspecies of tne D. obscuru group. The geographic origin of these strains and the source whence they were obtained are listed in table 1.
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VII
D. subobscura
15800 bp
Mvnl Hae III Hpall BamHI EcoRl Hind Ill Hpal Pst I Pvull Sacl Sea I Xbal
VIII D. guanche
15800 bp
Mvnl Hae Ill Hpall Eco RI Hind Ill Hpal Pvu II Sacl kl FIG. 1 (Continued)
mtDNA Extraction and Restriction Mapping mtDNA was obtained by the methods of Latorre et al. ( 1986) and Afonso et al. ( 1988)) with some modifications. The procedure yields an enriched fraction of mtDNA that gives well-resolved bands after restriction-enzyme digestion. Of the 13 restriction endonucleases used (bought from Boehringer Mannheim), 10 (BumHI, EcoRI, HindIII, HpaI, &I, PvuII, SacI, ScaI, XbaI, and XhoI) recognize 6-bp sequences; the other three (HaeIII, HpaII, and MvnI-Thai) recognize 4-bp sequences. The restriction fragments are separated on slab horizontal 0.8%-l .2% agarose gels, with TBE buffer (89 mM Tris-borate, 2 mM EDTA, pH 8 ). After electrophoresis, the gels are stained with ethidium bromide (0.5 ug/ml), and the DNA bands are visualized under UV light. A mixture of lambda phage DNA fragments obtained by single digestion with Hind111 and by double digestion with Hind111 and EcoRI is used as a standard to determine fragment size. Restriction-site maps are obtained by means of double digests. The homology among fragments of different species is verified by filter hybridization with digoxigeninlabeled probes (DIG DNA labeling kit; Boehringer Mannheim) of several mapped clones of D. yakuba (Clary and Wolstenholme 1985 ) . Results The 21 strains analyzed yield 16 different haplotypes, identified by roman numerals in table 1. All 14 taxa have distinctive haplotypes. The two strains of D.p. pseudoobscura and the two strains of D. algonquin have distinctive haplotypes, whereas only one haplotype is identified in each of the other three species represented by more than one strain. Elsewhere we have characterized a number of haplotypes from various
628
Barrio et al. Table 1 Geographic Origin of 21 Strains of 14 Taxa of Drosophila obscura Group
Strain,” Species afinis subgroup: l,D.a&is ................ 2,D.ajinis ................ 3, D. algonquin LI .......... 4, D. algonquin HF .......... 5, D. azteca ................ 6, D. azteca ................ 7, D. azteca ................ 8, D. azteca ................ 9, D. narragansett ........... 10, D. narragansett .......... II, D. tolteca ............... obscura subgroup: 12, D. subobscura ........... 13, D. guanche ............. 14, D. ambigua ............. 15, D. obscura .............. 16, D. bifasciata ............ pseudoobscura subgroup: 17, D. p. pseudoobscura M .... 18, D. p. pseudoobscura BC ... 19,D.p.bogotana .......... 20, D. persimilis ............ 21, D. miranda .............
Locality
mtDNA Haplotype
C&al Lake, Nebr. Lincoln, Nebr. Lincoln, Nebr. Honeoye Falls, N.Y. Davis, Calif. Chilpancingo, Mexico Arizona California Indiana Bastrop State Park, Texas Coroico, Bolivia
I I II III IV IV IV IV V V VI
Helsinki Tenerife, Canary Islands Ribarroja, Spain Queralps, Spain Akan-Ko, Japan
VII VIII IX X XI
Zirahuen, Mbico Bryce Canyon, Utah Bogotl Davis, Calif. Davis, Calif.
XII XIII XIV xv XVI
* 2, 5, and 9 were obtained from the University of Urn& (Sweden); 10 was from Dr. Luis Serra, and I5 wasfrom Dr. Maria Moncl6s, UniversidadCentral de Barcelona(Spain); I7 and I8 were from Dr. Wyatt W. Anderson, University of Georgia, Athens (USA); 7, 14. 20, and 21 were collected by Amparo Latorre.; and all other strains were obtained from the Drosophifucollection at Bowling Green State University, Ohio.
subgroup appears in 8 1% of cases; however, none of the nodes connecting the obscurasubgroup species approaches statistical significance. We have also obtained a maximum-parsimony unrooted tree that uses all the mtDNA restriction data available, including previously published information about the obscura group (Gonz6lez et al. 1990). The tree and the results of a bootstrap procedure ( 100 replicates) are similar to those obtained with our data alone: the pseudoobscura and a&is clusters are statistically validated, but none of the obscura nodes is (except for either the closely related pair D. subobscura and D. madeirensis or strains of a given species). We have used the matrix of cleavage sites to estimate the nucleotide divergence between haplotypes, following the maximum-likelihood procedure suggested by Nei ( 1987, p. 107). Table 3 shows the fraction of shared sites (see data above the diagonal) and the divergence per nucleotide when a molecular clock is assumed (see data below the diagonal). The means for the nucleotide divergence within and between clusters are given in table 4. The pattern that emerges is similar to the pattern in figure 3. When species are compared within each of the three subgroups, the a&is and pseudoobscura subgroups exhibit low average differentiation, whereas the obscura subgroup is much more heterogeneous. Indeed, the average genetic divergence between species within the obscura subgroup (0.10 1 f 0.0 14) is about the same as that between species
mtDNA
of Drosophila obscura
Phylogeny
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Table 2 Matrix of Presence-Absence of Phylogenetically Informative Restriction Sites in mtDNA of 16 Drosophila Haplotypcs STATUS OF HAPLOTYPEb SITES h4 ...... k3. ..... k4 ...... k5 ...... k? ...... k12 ..... kl7.. ... k23 ..... e3 ...... e4 ...... e7 ...... e9 ...... i2 ...... i3 ...... i5 ...... i7 ...... i8 ...... j2 ...... j3 ...... j4 ...... j6 ...... jl0 ..... vl ...... v2 ...... v3.. .... v4 ...... v5.. .... v6 ...... v7.. .... ~8.. .... sl ...... s2 ...... s3 ...... s5 ...... s7 ...... t4 ...... t5 ...... t7 ...... x2.. .... x5.. .... yl ...... y2 ...... y3 ......
I
II
000 000010 000 00 00 000 00 111110 111011 00 111110 111111 00 000 00 111111 111111 000101 111000 00 000011 00 111111 111011 111111 111010 00 111100 111111 100010 111111 111111 0110
00 111111 00 00 111110 111111 000 111010 111110 00
III
IV
v
00
0 0 0
0 0 0 0 0
0 0 0 0 0
VI
VII
VIII
IX
x
XI
XII
XIII
XIV
xv
XVI
0
0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 0 1 1 0 1 1 0 0 1 0 1 0 0 0 0 1 0 1 1 0 0 1 0 0 1 0 0 0
0 0 0 0 0 1 0 0 0 0 0 1
0
0 0
0 0 0
1 0 1 0 1 0 1 1 1 1 0 0 1
0 1 0 0
1 0 0 0 0 0 0
1 0 0 0 0 0 0
1 0 0 1 1 1 0 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0
1 0 0 1
1 0 1 0 1 0 1 1 1 1 0 0 1 1 1 0 0 0 0 0 0 1 0 0 1 1 1 0 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0
1 0 0 0 1 0
0 0 0
1 0 1 0 1 0 1 1 1 1 0 0 1 1
0 0 0 0 0 0 1 1 0 1 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 0 0 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0
0 0 0 0 0
0
0
0
0
0
0 0 0
0 0 0
0 0 0
0
0
0
0
0
1 0
0
10
0
0
0
0 0 0
0 0 0
11 0 0 0 0
00
0
0
0
0 0
0
0
1
1 0 0 0 1 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 0 0
0 0 1 0 0 0 0 0 0 0 1
0 1 0 0 0 0 1 0 0
1 0 0 1 0 1 1 0 0
1
0 1 0 0 1
0 1 0 1 1 1 0 1 0 0 1
1
0
1 0 1 1 1 0 1 0 0 1
0 0 0 1 1 0 0 0 1
0 0 0
0 0 0
0 0 1 0 1 0 0 0 1
0 0 1 0 1 1 0 0 0
1 0 0 1 1 1 1 0 1 0 0 0 0
1
1 1 0 0 0 0 0 0 1 1 0 1 0 1 0 1 0 0
1 1 1 1 0 0 1 1 1 0 0 0 0 0 0 1 0 0 1 1 1 0 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0
a Abbreviations for restriction enzymes are as in fig. 2. b 0 = Absence of phylogenetically informative site; and I = presence of phylogenetically informative site.
of different subgroups (0.105 f 0.0 12 for pseudoobscuru vs. @inis, 0.113 + 0,017 for pseudoobscura vs. obscura, and 0.097 f 0.011 for afinis vs. obscuru). To test for different mtDNA evolutionary rates in the obscura set of species, we
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FIG. 2.-Location and frequency of the restriction sites in the mtDNA of the Drosophila o~.rcuru group species. Each radial line corresponds to a restriction site; the length of the line is proportional to the percent of mtDNA haplotypes exhibiting the site. The mtDNA organization is based on the genetic map of D. yakuba (Clary and Wolstenholme 1985). The I I conserved sites are assigned by reference to sequence data (De Bruijn 1983; Clary and Wolstenholme 1985; Garesse 1988). The 93 variable sites are placed on the gene map on the basis of their relative positions on the restriction maps (see fig. 1) . b = BumHI; e = EcoRI; h = HueIII; i = HindIII; j = HpaI; k = HpaII; m = MvnI or Thai; p = MI; s = SacI; t = ScuI; v = PvuII; x = XbuI; and y = XhoI.
have applied the nonparametric test of Templeton ( 1983)) for which the restriction sites are the units of information. We have not obtained any statistically significant evolutionary-rate difference between any species or clusters of species (data not shown). Discussion Our data show that the traditional obscuru-afinis split of the obscuru group does not correspond well with the phylogenetic history of the group, which shows the Nearctic pseudoobscura species as a distinct subgroup, as different from the Palearctic obscuru species as are the species of the D. u&zis subgroup. The two Nearctic subgroups, pseudoobscura and afinis, are well-defined clusters, whereas the Palearctic obscuru subgroup consists of highly divergent species and is polyphyletic. The heterogeneity of the obscuru subgroup has been noted elsewhere (Cabrera et al. 1983; Latorre et al. 1988; Goddard et al. 1990; Gonzalez et al. 1990). Indeed, the degree of divergence among many of the obscuru subgroup species is as high as their divergence from the pseudoobscura and the ajinis subgroups.
mtDNA Phylogeny of Drosophila obscura
631
t ps. pseudoobscura BC c P E ,2 3 I 0
0
I 10
1
t
20
FIG. 3.-Unrooted phylogeny of the 16 haplotypes of the obscura group species, derived using Wagner’s parsimony method implemented on Felsenstein’s ( 1990) PHYLIP package, version 3.3. Gains and losses are weighted equally. The numbers on the branches and the scale on the abscissa refer to mutational steps. The numbers in the nodes are the frequency (in percent) with which a cluster appears in a bootstrap test of 100 runs, also implemented in the PHYLIP package. The three species subgroups are labeled on the right.
DeSalle et al. ( 1987) have discovered a “saturation effect” by comparing the percent nucleotide divergence between small sequenced regions of mtDNA with the time since the species divergence. It is known, from both sequence data and restrictionsite analysis, that there is a high percentage of bases A+T in the mtDNA of Drosophila species (De Bruijn 1983; Clary and Wolstenholme 1985; Wolstenholme and Clary 1985; DeSalle et al. 1987; Barrio et al. 1988; Garesse 1988). It is not known which selection pressures are responsible for this pattern, but it yields a saturation effect that reaches a plateau probably earlier than is the case for nuclear genes or for vertebrate mitochondrial genomes. DeSalle et al. ( 1987) have estimated that the saturation effect has important consequences whenever the compared Drosophila species exhibit >8% nucleotide differentiation, i.e., diverged >20 Mya. The saturation effect is apparent in the restriction-analysis reconstruction of the phylogeny of the obscura group. Let us assume, first, as a null hypothesis, that there is no saturation effect. We can estimate divergence time by applying to our data (table 3) either one of two evolutionary rates: 1.7% (Caccone et al. 1988) or 0.5% (Latorre et al. 1988) sequence divergence per Myr. The time for the divergence of the D. miranda lineage from the other pseudoobscura species would be 0.8 or 2.6 Mya, respectively. These dates are consistent with the postulated association between the Pleistocene glaciations (2 million-20,000 years ago) and Drosophila speciations in temporarily isolated refuges (Dobzhansky and Powell 1975 ). However, the same rates give 3 or 11 Mya as the time of divergence of the three obscura subgroups. These dates are 29-43 Myr more recent than the timing for the melunoguster-obscuru split, which, on the basis of biogeographic considerations, occurred before the mid-Oligocene (Throckmorton 1975) and, according to molecular data, -40 Mya (Sharp and Li
Table 3 Fraction of Shared Sites (above Diagonal) and Divergence per Nucleotide (below Diagonal), between mtDNA haplotypes of Drosophila obscura-Group Species HAPLO~PE HAPLOTYPE,SPECIES I, a&is II, algonquin LI III, algonquin HF IV, azteca V, narragansett VI, tolteca VII, subobscura VIII, guanche IX, ambigua X, obscura XI, bifasciata XII, p. pseudoobscura M XIII, p. pseudoobscura BC XIV, p. bogotana XV, persimilis XVI, miranda
I
0.013 0.010 0.027 0.023 0.051 0.106 0.109 0.085 0.114 0.094 0.104 0.108 0.107 0.097 0.098
II
III
IV
v
VI
VII
VIII
IX
x
XI
XII
XIII
0.930
0.943 0.986
0.862 0.875 0.889
0.877 0.889 0.901 0.818
0.754 0.794 0.806 0.807 0.771
0.563 0.571 0.581 0.561 0.646 0.590
0.554 0.594 0.603 0.552 0.606 0.613 0.632
0.627 0.636 0.646 0.633 0.618 0.656 0.610 0.600
0.546 0.553 0.560 0.543 0.564 0.568 0.551 0.571 0.667
0.603 0.583 0.592 0.606 0.595 0.600 0.523 0.606 0.588 0.539
0.571 0.580 0.588 0.540 0.592 0.508 0.516 0.571 0.554 0.480 0.620
0.559 0.567 0.576 0.525 0.580 0.492 0.500 0.557 0.540 0.466 0.580 0.939
0.003 0.024 0.021 0.041 0.103 0.095 0.082 0.111 0.100 0.100 0.105 0.104 0.094 0.094
0.021 0.018 0.038 0.100 0.092 0.080 0.108 0.097 0.098 0.102 0.101 0.091 0.091
0.036 0.039 0.107 0.110 0.084 0.115 0.093 0.115 0.121 0.118 0.108 0.097
0.047 0.080 0.092 0.088 0.107 0.097 0.097 0.101 0.100 0.091 0.082
0.097 0.090 0.077 0.106 0.095 0.126 0.133 0.130 0.120 0.110
0.086 0.093 0.114 0.124 0.126 0.132 0.129 0.118 0.108
0.096 0.107 0.095 0.106 0.111 0.109 0.099 0.099
0.077 0.100 0.111 0.117 0.115 0.104 0.094
0.119 0.141 0.148 0.144 0.135 0.116
0.091 0.104 0.094 0.085 0.094
0.012 0.008 0.006 0.028
0.015 0.012 0.037
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Table 4 Mean and Standard Deviation (SD) for Nucfeotide Divergence Within and Between Sets of Species
Species (no. of comparisons) afinis subgroup (15) ............................... pseudoobscura subgroup ( 10) ........................
obscura-subgroup ( 10) .............................. obscura-ambigua cluster (1) ......................... subobscura guanche cluster (1) ....................... pseudoobscura vs. afinis subgroups (30) ............... pseudoobscura vs. obscura subgroups (25) .............. afinis vs. obscura subgroups (30) ..................... pseudoobscura vs. obscura-ambigua (10) ............... aJinis subgroup vs. obscura-ambigua ( 12) .............. pseudoobscura subgroup vs. subobscura-guanche (10) ..... a&is subgroup vs. subobscura-guanche (12) ............ pseudoobscura subgroup vs. bifasciata (5) .............. afinis subgroup vs. bifasciata (6) ..................... obscura-ambigua vs. subobscura-guanche (4) ............ obscura-ambigua vs. bifasciata (2) .................... subobscura-ambigua vs. bifasciata (2) .................
Mean + SD Substitutions per Site 0.027 + 0.013 0.018!I 0.010 0.101 f 0.014 0.077 + 0.000 0.086 f 0.000 0.105 + 0.012 0.113 + 0.017 0.097 f 0.0 11 0.122 + 0.017 0.096 f 0.014
0.114+0.011 0.098 f 0.009 0.094 + 0.006 0.096 f 0.002 0.102k 0.009 0.110f 0.009 0.109 + 0.014
1989) or 46 mya (Beverley and Wilson 1984). The evidence indicates that the divergence of the obscura lineages occurred shortly after the melanogaster-obscura split, in association with the expansion of deciduous forest throughout the Palearctic region during the mid-Oligocene. An early split of the obscura lineages, shortly after the obscura-melanogaster divergence, is also supported by our observation (data not shown) that the melanogaster mtDNA clusters within the obscura group set of lineages. The discrepancy between the mtDNA divergence dates for the obscura subgroups and the dates estimated from other information could be explained by postulating that the evolution of the mtDNA in all obscura lineages virtually stopped between 30 and 2 Mya-something for which no other support exists. A more likely alternative explanation for the discrepancy is the saturation effect described above. The bias favoring A+T in Drosophila mtDNA not only entails an apparent reduction in evolutionary rate as time becomes remote, but it also diminishes the discriminatory power that mtDNA restriction analysis has for reconstructing the phylogeny of species not closely related. Most restriction enzymes recognize sequences containing at least one G and one C. These bases are not abundant in the mtDNA of Drosophila; they are mainly located in functionally important positions of the rRNA genes and in the first- and second-codon positions of protein-coding genes. The scarcity of G+C entails a low number of restriction sites, such as is typically observed, in the mtDNA of Drosophila. The variability of the restriction sites in protein-coding genes is mainly due to changes in third-base positions, which are biased toward A or T, as has also been shown by codon usage (De Bruijn 1983; Wolstenholme and Clary 1985; Garesse 1988 ). The bias toward A or T also entails a high degree of homoplasy in the restriction sites shared between phylogenetically distant species. Acknowledgments
This work has been supported by a “Conselleria de Cultura, Educaci6n y Ciencia de la Generalitat Valenciana” fellowship to E.B., by grants PB90-0429 of DGICYT
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and BI089-0668 of CICYT (Spain) to A.M., and by U.S. Department of Energy grant ER607 13 to F.J.A. LITERATURE CITED AFONSO,J. M., J. PESTANO,and M. HERNANDEZ. 1988. Rapid isolation of mitochondrial DNA from Drosophila adults. Biochem. Genet. 26:38 l-386. BARRIO, E., A. MOYA, M. PEREZ-ALONSO,and A. LATORRE. 1988. Functional role of the GC sequences in the Drosophila mitochondrial DNA. Genet. Life Sci. Adv. 7:149-153. BEVERLEY,S. M., and A. C. WILSON. 1984. Molecular evolution in Drosophila and the higher Diptera. II. A time scale for fly evolution. J. Mol. Evol. 21: 1-13. BUZZATI-TRAVERSO, A., and R. E. SCOSSIROLI.1955. The obscura group ofthe genus Drosophila. Adv. Genet. 7:47-92. CABRERA,V. M., A. M. GONZALEZ,J. M. LARRUGA, and A. GULLON. 1983. Genetic distance and evolutionary relationships in the Drosophila obscura group. Evolution 37:675-689. CACCONE, A., G. D. AMATO, and J. R. POWELL. 1988. Rates and patterns of scnDNA and mtDNA divergence within the Drosophila melanogaster subgroup. Genetics 118:67l-683. CARIOU, M. L., D. LACHAISE,L. TSACAS,J. SOURDIS,C. KRIMBAS,and M. ASHBURNER. 1988. New African species in the Drosophila obscura species group: genetic variation, differentiation, and evolution. Heredity 61:73-84. CLARY, D. O., and D. R. WOLSTENHOLME.1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J. Mol. Evol. 22: 252-27 1. DE BRUIJN, M. H. L. 1983. Drosophila melanoguster mitochondrial DNA, a novel organization and genetic code. Nature 304:234-24 1. DESALLE, R., T. FREEDMAN,E. M. PRAGER, and A. C. WILSON. 1987. Tempo and mode of sequence evolution in mitochondrial DNA of Hawaiian Drosophila. J. Mol. Evol. 26:157164. DESALLE, R., and L. V. GIDDINGS. 1986. Discordance of nuclear and mitochondrial DNA phylogenies in Hawaiian Drosophila. Proc. Natl. Acad. Sci. USA 83:6902-6906. DOBZHANSKY,TH., and J. R. POWELL. 1975. Drosophila pseudoobscura and its American relatives, Drosophila persimilis and Drosophila miranda. Pp. 537-587 in R. C. KING,ed. Handbook of genetics, vol. 3: Invertebrates of genetic interest. Plenum, New York. FELSENSTEIN,J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-79 1. 1990. Phylip manual, version 3.3. University Herbarium of the University of California, -. Berkeley. GARESSE,R. 1988. Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations. Genetics 118:649-663. GODDARD, K., A. CACCONE, and J. R. POWELL. 1990. Evolutionary implications of DNA divergence in the Drosophila obscura group. Evolution 44: 1656- 1670. GONZALEZ, A. M., M. HERNANDEZ,A. VOLZ, J. PESTANO,J. M. LARRUGA,D. SPERLICH,and V. M. CABRERA. 1990. Mitochondrial DNA evolution in the obscura species subgroup of Drosophila J. Mol. Biol. 31: 122- 13 1. HALE, L. R., and A. T. BECKENBACH.1985. Mitochondrial DNA variation in Drosophila pseudoobscura and related species in Pacific Northwest populations. Can. J. Genet. Cytol. 27: 357-364. HARRISON, R. G. 1989. Animal mitochondrial DNA as a genetic marker in population and evolutionary biology. Trends Ecol. Evol. 4:6- 11. LAKOVAARA,S., and L. KER.&NEN.1980. Phylogeny of the Drosophila obscura group. Genetika 12:157-172. LAKOVAARA,S., and A. SAURA. 1982. Evolution and speciation in the Drosophila obscura group. pp. l-59 in M. ASHBURNER,H. L. CARSON,and J. N. THOMPSON,eds. The genetics and biology of Drosophila. Vol. 3b. Academic Press, New York.
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editor
Received July 19, 1991; January 13, 1992 Accepted January 13, 1992
