J Mol Evol (1990) 31:122-131

Journal of Molecular Evolution (~ Springer-Verlag New York Inc. 1990

Mitochondrial DNA Evolution in the obscura Species Subgroup of Drosophila

Ana M. Gonzfilez, 1 Mar iano Hernfindez, ~ Andrea VOIz, 2 Jos6 Pestano, 1 Jos6 M. Larruga, 1 Die ther Sperlich, 2 and Vicente M. Cabrera ~

' Department of Genetics, University of La Laguna, Canary Islands, Spain 2 Lehrstuhl rtir Populationsgenetik, Universit~it Tiibingen, Federal Republic of Germany

Summary. Mitochondria l D N A (mtDNA) restric- t ion site maps for nine species o f the Drosophila obscura subgroup and for Drosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total o f 105 different sites were detected, which corresponds to a sample of 3.49% of the m t D N A genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or vari- able rates o f evolution. Both methods led to the same relationships. Five differentiated clusters were found for the obscura subgroup species, one Nearc- tic, represented by Drosophila pseudoobscura, and four Palearctic, two grouping the related triads o f species Drosophila subobscura, Drosophila madei- rensis, Drosophila guanche, and Drosophila ambi- gua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each, Dro- sophila bifasciata, and Drosophila tristis. The dif- ferent Palearctic clusters are as distant between themselves as with the Nearctic one. For the related species D. subobscura, D. madeirensis, and D. guanche, the pair D. subobscura-D, madeirensis is the closest one. The relationships found by nucleo- tide divergence were confirmed by differences in mi tochondr ia l genome size, with related species sharing similar genome lengths and differing f rom the distant ones. The total m t D N A size range for the obscura subgroup species was from 15.5 kb for D. pseudoobscura to 17.1 for D. tristis.

Key words: Drosophila obscura subg roup --

Offspring requests to: A.M. Gonzfilez

Mitochondria l D N A -- Restr ict ion maps -- Phy- logeny

Introduction

The obscura group o f the genus Drosophila was thor- oughly reviewed at the morphological and chro- mosomal levels in 1955 by Buzzat i -Traverso and Scossiroli with the hope o f offering a unified picture of the phylogenetic relationships between Eurasiatic and Amer ican members o f the group. The conclu- sions the authors reached were that affinis and ob- scura subgroups differ f rom one another more than species o f the same subgroup belonging to different zoogeographical regions, and that, in the obscura subgroup, species have diverged more between zoo- geographical regions than within them and are rep- resented by one complex in the Nearctic and several complexes in the Palearctic. Since then, new species o f the group have been discovered or rediscovered, including the Atlantic island endemisms o f Dro- sophila guanche (Monclfis 1976) and Drosophila madeirensis (Monclfas 1984) and the African species Drosophila microlabis, Drosophila kitumensis, Dro- sophila krimbasL and Drosophila cariouae (Tsacas et al. 1985). Numerous new phylogenetic studies have been carried out, based mainly on electropho- retie compar isons (Lakovaara et al. 1972, 1976; Marinkovi6 et al. 1978; Pinsker and Buruga 1982; Cabrera et al. 1983; Loukas et al. 1984; Cariou et al. 1988). There have been fewer studies o f the group at the genomic D N A level (Loukas et al. 1986). Despite these data, some ambiguities persist in the

r e l a t i o n s h i p s b e t w e e n these species . T h e c lass ica l s u b d i v i s i o n o f t h e obscura s u b g r o u p i n to two c lus - ters, one wi th the t h ree N e a r c t i c spec ies a n d the o t h e r wi th all o f the P a l e a r c t i c spec ies has been re- p e a t e d l y q u e s t i o n e d ( F a r r i s 1974; M a r i n k o v i 6 el al. 1978; C a b r e r a et al. 1983; L a t o r r e et al. 1988). S i m - i lar ly, t h e r e is n o a g r e e m e n t o n the r e l a t i v e p a i r i n g o f the r e l a t e d t r i a d o f P a l e a r c t i c spec ies Drosophila subobscura, D. guanche, a n d D. madeirensis (Ca- b re ra et al. 1983; L a r r u g a a n d P i n s k e r 1984; L o u k a s et al. 1984; K r i m b a s a n d L o u k a s 1984; C a r i o u et al. 1988; B a c h m a n n et al. 1989). R e c e n t l y , m i t o - c h o n d r i a l D N A ( m t D N A ) has been u sed as a t oo l for p h y l o g e n e t i c in fe rence , e spec i a l l y a m o n g c lose ly re la ted t a x a ( r e v i e w e d in W i l s o n et ai. 1985; A v i s e 1986; M o r i t z e t al. 1987). A p a r a d i g m a t i c s t u d y exists for Drosophila in w h i c h d e t a i l e d r e s t r i c t i o n m a p p i n g o f m t D N A has p r o v i d e d a v e r y a c c u r a t e p h y l o g e n e t i c t ree for e igh t spec i e s o f t he melano- gaster s u b g r o u p (So l ignac et al. 1986a). C o r r e s p o n d - ing d a t a o n the obscura s u b g r o u p a re v e r y l i m i t e d , being r e s t r i c t e d to two s t ud i e s b a s e d o n r e s t r i c t i o n f r agmen t c o m p a r i s o n s ( H a l e a n d B e c k e n b a c h 1985; La to r r e et al. 1988).

In the p r e s e n t s t u d y w e h a v e t r i e d to d e t e r m i n e the p h y l o g e n y o f n ine D. obscura s u b g r o u p species , i nc lud ing o n e N e a r c t i c a n d e igh t Pa l ea r c t i c species , Using d e t a i l e d r e s t r i c t i o n s i te m a p s in o r d e r to c o m - Pare t h e i r r e l a t i o n s h i p s a t t he m t D N A leve l w i th those p r e v i o u s l y e s t a b l i s h e d b y o t h e r m e a n s .

Hpa I d i g e s t s

P r o b e 1

P r o b e 2

123

Materials and Methods

Species and Strains. Nine D. obscura subgroup species and D. melanogaster (an outgroup species) were used in this study. For each of the 10 species at least 2 isofemale strains were analyzed. The species and numbers of strains were as follows:

Drosophila melanogaster: cosmopolitan species of the melano- gaster subgroup, two strains from Giiimar (Canary Islands), which represent the two more common mtDNA morphs in a sample of 100 strains, have been chosen.

Drosophila subobscura: four strains representing the more abun- .dant and cosmopolitan mtDNA morphs of the species detected m previous worldwide screens (Afonso et al. 1990; Rozas et al. 1990).

Drosophila guanche: endemic of the Canary Islands, 13 strains caught in a laurel forest in Tenerife Island.

Drosophila madeirensis: endemic of Madeira, three strains also caught in a laurel forest.

Drosophila ambigua: seve~ strains total: four strains caught in a mixed forest of oak and pine in Escorial (Central Iberian Pen- insula), one strain from Tiibingen (Germany), one strain from northern Italy, and one strain from Rochefort (France).

Drosophila pseudoobscura: the only Nearctic representative, eight strains from laboratory stocks of the Molecular Biology Center of Madrid (two), Kyushu University (two), and Genetics de- partments of the Tiibingen (two) and Barcelona (two) univer- sities.

Drosophila bifasciata, Drosophila obscura, Drosophila subsilves- tris, and Drosophila tristis: two independent strains for each

Fig. 1. Top, ethidium bromide-stained agarose gel with the HpaI mitochondrial DNA patterns for the nine Drosophila ob- scura subgroup species (h, lambda DNA digested with HindlII; T, D. tristis; B, D. bifasciata; V, D. subsilvestris; 0, D. obscura; A, D. ambigua; G, D. guanche; M, D. madeirensis; S, D. sub- obscura; P, D. pseudoobscura). DNA from this gel was transferred to a nylon membrane and sequentially hybridized with pDYHC (probe 1) and 62F9 (probe 2). The two main DNA fragments of D. obscura hybridized with probe 1, but only the larger one with probe 2. There are small bands with similar migration for D. obscura. D. ambigua, and D. guanche species but only that of D. ambigua hybridized with probe 1.

of these Palearctic species obtained from laboratory stocks of the Genetics departments of Tiibingen and Barcelona.

Mitochondrial DNA Preparation and Mapping Procedures. Mi- tochondrial DNA isolation from adult flies, restriction enzyme digestions, electrophoresis, and the recording of fragment length patterns have been previously described (Afonso et al. 1988; Rozas et al. 1990). Twelve restriction enzymes were used, 2 (Hae- III and HpaII) recognize 4-bp sequences and 10 (BamH1, EcoRI, EcoRV, HindIII, HpaI, Pstl, PvuII, SacI, XbaI, and XhoI) rec- ognize 6-bp sequences.

Restriction site maps were constructed in three stages: double digestions with pairs of enzymes that give only a few restriction fragments, partial digestions for enzymes giving numerous and/

124

Table 1. Observed and expected frequencies of restriction sites per mitochondrial genome, and the total number observed for all the species

Endo- Mean sites per genome Total

nucleases Observed Expected sites

Table 3. Mitochondrial DNA sizes and intra- and interspecific mean values (as percentages) for the 10 species

Mean 6 values (%) Mitochon- drial DNA Intra- Inter-

Species size (bp) specific specific

BamHI 0.20 4- 0.42 0.36 2 EcoRl 4.40 + 0.70 4.48 9 EcoRV 1.81 + 0.87 4.48 8 HindlIl 3.83 4- 0.58 4.48 11 HpaI 2.40 4- 1.17 4.48 10 PstI 0.50 4- 0.85 0.36 3 PvulI 3.20 4- 1.48 0.36 9 SacI 3.70 4- 1.06 0.36 7 XbaI 2.55 4- 0.93 4.48 8 XhoI 0.30 4- 0.48 0.36 2

HaelII 2.94 4- 1.53 2.34 17 HpalI 4.64 4- 1.15 2.34 20

Total 30.47 28.88 106

D. pseudoobscura 15,500 0.9 _+ 0.5 13 + 1 D. subobscura 15,900 0.8 + 0.4 10 _+ 3 D. madeirensis 15,900 12 + 3 D. guanche 15,900 11 + 2 D. ambigua 16,100 1.0 + 0.4 10 + 2 D. obscura 16,1 O0 11 +4- 3 D. subsilvestris 16,100 10 -2_ 2 D. bifasciata 16,500 12 + 1 D. tristis 17,100 12 + 2 D. melanogaster 18,700 0.5 + 0.4 12 + 2

Total mean 0.8 + 0.1 11.3 4- 0

Table 2. Restriction patterns for nucleomorphs ofpolymorphic species

Nucleomorphs EcoR V HindlII XbaI HaeIlI HpaIl

D. subobscura-2 A B A B B D. subobscura-3 A A A C A D. subobscura-8 A B A C A D. subobscura- 13 A A A A A

D. ambigua-1 A A A A A D. ambigua-2 A A B B A D. ambigua-3 B A B C B

D. pseudoobscura- 1 A A A A B D. pseudoobscura-2 A A A A A D. pseudoobscura-3 A A A B C

D. melanogaster- 1 A A A A A D. melanogaster-2 A B A B A

or small fragments, and tests of fragment homology among species by filter hybridization with the already mapped clones of D. melanogaster 62F9, H, B, and M3 (Garesse 1988) and of Dro- sophila yakuba pDYHB, pDYHC, and pDYHD (Clary and Wol- stenholme 1985). DNA from clones was obtained using the large- scale plasmid DNA isolation method of Maniatis et al. (1982) and was photobiotinylated as described in Forster et al. (1985) for use as hybridization probes. Gels were stained with ethidium bromide (Maniatis et al. 1982), photographically recorded, trans- ferred to nylon membranes (Biodyne) (Southern 1975), and se- quentially hybridized (Johnson et al. 1984) with different probes to cover the whole mtDNA molecule. The resolution of the meth- od is shown in Fig. 1. Restriction fragment sizes were determined using lambda DNA cut with HindllI and pBR322 DNA cut with HaellI as reference markers, using the Schaffer and Sederoff(1981) program for length estimation. Because the estimation of the length of large DNA fragments is imprecise, size estimates were therefore based on homologous fragments already sequenced in D. yakuba (Clary and Wolstenholme 1985).

Restriction Pattern Nomenclature. The different restriction pat- terns of a given enzyme were designated by capital letters, ac- cording to Afonso et al. (1990), and the composite morphs (nu-

cleomorphs) deduced from the patterns of all enzymes together (Nei and Tajima 1981) were denoted by Arabic numbers.

Results

Mitochondrial DNA Polymorphism and Restriction Site Maps

The mean number o f restriction sites observed per m t D N A molecule and the total number found in this analysis are given in Table 1 and compared with the expected number assuming a m t D N A size o f 16 kb, a r andom nucleotide distribution, and a fre- quency o f G + C o f 0.22 (Kaplan and Langley 1979). There are significant differences between expected and observed restriction sites (X / = 59.43; d f = 11; P < 0.001), mainly due to the excess o f sites ob- served with the enzymes PvuII and SacI.

Taking into account all restriction enzymes (12) and strains (45) analyzed, a total o f 105 different restriction sites have been detected, which corre- sponds to a sample o f 3.49% of the mi tochondr ia l genome. Only nine o f these sites were identical in all strains. On average 30 _+ 3.5 sites were mapped per strain, which is about 1% o f the mi tochondr ia l genome.

The restriction site maps established for the 10 species and their po lymorphic variants are pre- sented in Fig. 2. The molecule was linearized at the conserved XbaI site close to the beginning o f the coding region, such that the A + T - r i c h region was at the f ight-hand end o f the map, as in Solignac et al. (1986a). The mi tochondr ia l genome organization is also shown in Fig. 2. Our methods o f mapping did not allow us to detect restriction fragments smaller than 500 bp with e th id ium bromide and UV light, nor smaller than 300 bp with photobi- ot inylated probes. Restr ict ion sites for the same en- zyme differing in map location by less than 200 bp

O + l U h O b I c u I a 15900 bp

- - ~ _ . ~ ,3o0 I 0o0o

~ o o o o 150i~ I | 900 ~ 4810 90OO

15900 8450 J 5300 J2150

3700 5300 2150 - - _ . , _ ] , 0 0 I 75oo I

8250 ~ 6800 15000 ooo I OlOO I 550o

14800 rl3oo Ps' t , Ps ' t l Ba~HI

EcoR, EcoRV H a e , I I ' k H n e , I , . D H a ~ H I . i I I , . A H I . o l I I + B Hpl Hpi I . A Hpa I . B Pro , Sac Xbl

II'mad al r a n t i s 15000 bp

~ , , 3 0 0 r 2 0 o 0 I 05no EccR, oSTOO 122001 [�9 nv

14900 H i e l l l o I 5300 12150 H I n H I , ,

i 15900 Hpa, % 1 3 4 5 0 0 1 4 8 0 0 ii~ili05pi 4550 Hpa'lpvu,,

~ 0 1 5 o 5 5 0 9 1 0 2 0 5 0 o 1 280o i 8000 Sac, X b l l

P s~'~-I Pa'I I

I1' ambl DUC 15100 bp ~ , o 0 I 055o 12 ,oo l 305o EcoR,

8700 J 7400 EcoRV+B 0 7000 H o e , , I - A

7000 H . c I , I " D 555o 7oo0 Hal I 1 , . C

~ ~ 5 o I 5300 J5350 H i n d , I , 7,o I,,5ol ,o.,

88115098100 850 o Hpa , , �9 A 15oo I 5300 H p a , , . B i 285o ~ 0o5o P v u l ,

, :3oo I 0'1oo I 0":,00 s . ~ l 080000 I 3058~176176176176 Xlal.Xba' AD

xh'o,

i l l t a U d o c i o s c u r i 1540000

~ ~ _ _ _ _ , 300 l 0200 EcoRI

~ 2o00 12 119 ~ ~176176 t c o n v 700 Ha~ 1,1" A

07o0 14000 I l l e l l l ' n ~ o 115oo[ 85oo H i n d , , I

~ 0 0 15500 I " , . . I ,+oo II79OI , n o o Hp. II A I , . oo 1.001,0o0 . , . , , .

~ 0 ~I 2500 J1350 . 0 0 , 5 0 0 H p n l I . C ""--------___.~.]~a5D 1 2 5 5 o I ~ P v . , ,

123001 2000 l S l c n oo J COOl X o . ,

Xhol

O'i~e I In a l a s t er 10700 bp

~ ~ ' 5 ' 00 117001 10700 t + * n l

~ oo I [+o nv 5400 imol 005o H a e , l l . A

0100 i 880o H n e l l I . B (~1 4750 I , 050 H I n d I , I . A

4750 4000 [ H i n d , I t ' D

~ 1 11 0350 15790*55o . , i t 3650 H p i ' ' 00 ] 2 O 0 oleeollOr~l 11300 P v . I I

~ ~ , 0 o 0 0 0 I 5,95 +510905o0 +.c, ItO01~ X b . I

Ps'I , Xh'~ I

| . I man eke 111119p

JIt~1111~ i s l e I o l e o t l e l l 14,,, n t , o .

I l l l l l i n . I l l I nose I|I 1241 12111 l l l n d l l l

1 4 + . 0 11,,81 11,oo i , , l I 5110 ~l i~ Oleo I 4440 I101 I I

I l l l l l l l l l P v . J I 12208 n = , , , l .8001 2o0o i O i l , l o l l

i 14ooo 1ore i gm l

| . l i l l l r l 11111 ill I I ~ 1411 I 41111 + 4 l l l l [ l , l l

I 11111 11411 E,:, IV I , l i e I 1311 1111181 , l i e M , , I I I

I 0 , o 0 I o2501 8111 12948 N l a I I , I 7 , 5 , 11oo,1 8 , 0 , 10401 . 0 , e

I 9050 I~l 2059 11ooo]il 4158 N I , * I I

I ? , " i I . . 00 , , . , , ] l J 0710 l l C J I l O l l I 4111 11111 f i l l

O. l U b l l I v a i l r Is 1610000

luool 5,00 I 5500 I 3550 EcoRI 15100 r t ca illf

[ 1120o I 405o H a ~ I 5050 115001 5800 12550 H i , d i l l

eeoo ! 7500 I Hpa I 5300 I 50012100110501 . 5 0 0 0 . , . l .

I 3 1 5 ~ I 3 5 0 0 11.01 7600 F l u ' ' I +000 l 5lOO I 70oo s . , ,

I 0 0 0 0 I 0 0 0 0 11400 x o , I

O. 9 l I | ICJI~ I I 10509 bp

~1mo~11o0[ s900 ~,~ 50oo I , 3 5 o Er 10590 I Eta RV

I , , 5 0 i 2 5 , 0 l 2700117091 ,590 , . . l , I 1 8 0 5 0 115001 5 3 0 0 1 2 7 5 0 H , . + l S t

I 2550 118ool 12000 Hpal I 0950 ] 8609 H 5550 H , , I I

1855O 1 2 8 5 0 I P v u l i I + 8 0 0 1 0 8 0 0 1 5000 IPl 0000 s , ~ l

I 0 8 0 9 i 8 0 0 0 x b , ,

O. 1 r i l l Is 1710000

1000i1100 8390 i f 0090 E�9 3050 4256 | 0866 Ec~RV

11500119501005012soo l ,o~ 7300 I ( a l l l l I 0050 115001 5090 I 0350 e l . o i l l

1205o,,0o0 ,0o0 i 0 o o o , , , , I 5 5 5 0 ~ I~1 l a D 0 ~ 8 1 0 0 I lp l I I

I 51oo I 2550 J1051~ 10050 P . u l l 12000 I 5100 l S l c l

l 0859 I 5559 113001 5150 x o , i | l 'mHI

0 I 10 18 r l i l l i l r l J i l l l l P kb

125

Fig. 2. Mitochondrial DNA site restriction maps of the l 0 Drosophila species studied. The BamHl, Pstl, and Xhol sites are indicated Under the corresponding maps. Intraspecific patterns for polymorphic restriction sites are shown in consecutive lines. The equivalent genome organization, at the bottom, is taken from Clary and Wolstenholme (1985).

126

TaMe 4. t~ +_ SE values for pairs of nucleomorphs (multiplied by 103)

Nucleo- morphs* P- 1 P-2 P-3 S-2 S-3 S-8 S- 13 M G

P o l

P-2 3 + - 3 -- P-3 10 +- 6 13 _+ 7 -- S-2 123 -+ 30 119 _+ 29 134 4- 31 -- S-3 116 +__ 29 112 _+ 28 128 +_ 30 14 _+ 7 -- S-8 119 + 29 115 + 28 131 --+ 31 11 +- 6 4 +- 4 -- S-13 120 --- 29 116 -+ 29 131 +- 31 11 +- 6 4 __- 4 7 - 5 M 127 + 30 123 + 30 138 -+ 32 58 + 17 42 --- 14 45 + 15 G 123 + 29 119 +_ 29 134 + 31 95 + 24 88 +_ 23 91 + 24 A-I 122 +_ 28 119 +_ 28 133 + 30 107 + 26 100 _+ 25 103 --- 25 A-2 116 _+ 28 112 • 27 127 _ 29 100 + 25 93 __- 24 97 + 24 A-3 124 +- 29 120 + 28 135 +- 30 108 + 26 102 ___ 25 105 +_ 26 O 138 +_ 30 134 + 30 136 +_ 29 111 _ 26 105 _+ 25 107 - 25 V 114 +_ 28 110 +_ 27 114 + 27 88 +_ 23 81 • 22 84 +_ 22 B 116 +_ 27 112 -4- 27 126 +- 28 136 -4- 31 117 • 28 120 --- 28 T 131 • 29 127 + 28 130 + 28 152 +- 33 145 • 32 148 + 32 na-1 145 +-- 32 141 +- 31 156 +_ 33 117 +_ 27 111 _+ 26 114 +_ 26 m-2 1 4 4 + 3 2 141 + 3 1 1 5 5 • 1 1 6 • 1 1 0 • 113+-26

Nucleo- morphs A- 1 A-2 A-3 O V

m

42 _+ 14 -- 92 _+ 24 88 + 23 --

104 _+ 25 135 _+ 31 119 + 28 97 _+ 24 128 +- 30 112 _+ 27

105 _+ 26 137 +_ 31 120 _+ 28 108 +_ 25 150 +_ 33 I l l -4- 26 84 +- 22 102 _+ 25 l l 0 _+ 27

121 _+ 28 128 +_ 29 124 _+ 29 149 + 32 155 + 33 127 +_ 28 114 +_ 27 132 _+ 30 129 -4- 29 1 1 4 +- 26 132 +_ 29 128 + 29

B T m - 1 m - 2

m - I

A-2 6 + _ 4 -- A-3 12 +_ 6 12_+ 6 -- O 66 +_ 17 61 +_ 16 75 _+ 19 V 80 -+ 20 73 +_ 20 81 _+ 21 B 112 +_ 26 106 _ 25 114 +- 26 T 116 -+ 25 i I 1 _+ 25 98 + 22 m-1 79 +- 19 73 +- 19 71 +_ 18 m-2 87 +- 21 81 _+ 20 79 +_ 19

58 _+ 16 - -

1 0 6 _ 24 127 _+ 29 - -

i 10 4- 24 109 _ 25 101 _+ 23 - -

1 1 0 +_ 24 99 • 24 144 -+ 31 106 _+ 23 - -

l l 0 +- 24 99 • 23 143 -+ 31 115 _+ 25 5 +- 4

* Abbreviations as in Fig. 3, m = D. melanogaster

i n d i f f e r e n t s p e c i e s h a v e b e e n a s s u m e d t o b e i d e n -

t i ca l .

A t o t a l o f 18 d i f f e r e n t n u c l e o m o r p h s h a v e b e e n

s t u d i e d , i n c l u d i n g t h o s e i n D. subobscura (4) a n d D.

melanogaster (2) d e t e r m i n e d p r e v i o u s l y ( A f o n s o e t

a l . 1 9 9 0 a n d u n p u b l i s h e d r e s u l t s ) . I n t r a s p e c i f i c p o l y -

m o r p h i s m s h a v e a l s o b e e n d e t e c t e d i n D. pseu- doobscura (3) a n d D. ambigua (3) . P o l y m o r p h i c r e -

s t r i c t i o n p a t t e r n s f o r t h e a p p r o p r i a t e e n z y m e s , t a k e n

f r o m Fig . 2, a r e g i v e n i n T a b l e 2 f o r e a c h n u c l e o -

m o r p h .

I n t e r s p e c i f i c v a r i a b i l i t y i n t h e o v e r a l l l e n g t h o f

m t D N A w a s a l s o o b s e r v e d ( T a b l e 3). A s n o t e d p r e -

v i o u s l y ( H a l e a n d B e c k e n b a c h 1 9 8 5 ; S o l i g n a c e t al .

1 9 8 6 b ) , t h i s l e n g t h v a r i a t i o n w a s d u e t o d i f f e r e n c e s

i n t h e A + T - r i c h r e g i o n .

Nucleotide Distances and Phylogenetic Trees

T h e d e g r e e o f n u c l e o t i d e d i v e r g e n c e b e t w e e n t h e

d i f f e r e n t s t r a i n s a n d s p e c i e s w a s c a l c u l a t e d , a s ~ v a l -

ues , f o l l o w i n g t h e m a x i m u m l i k e l i h o o d m e t h o d o f

N e i a n d T a j i m a ( 1 9 8 3 ) f o r t h e e s t i m a t i o n o f t h e

n u m b e r o f n u c l e o t i d e s u b s t i t u t i o n s f r o m r e s t r i c t i o n

s i t e d a t a . T h e m a t r i x o f d a t a is s h o w n i n T a b l e 4.

M e a n s o f i n t r a s p e c i f i c a n d i n t e r s p e c i f i c d i s t a n c e s o f

e a c h n u c l e o m o r p h o r s p e c i e s w i t h a l l t h e o t h e r s a r e

g i v e n i n T a b l e 3. I t c a n b e s e e n t h a t t h e m e a n i n -

t e r s p e c i f i c d i v e r g e n c e ( I 1 .3%) is a n o r d e r o f m a g -

n i t u d e g r e a t e r t h a n t h e i n t r a s p e c i f i c d i v e r g e n c e

( 0 . 8 % ) . T h e m e a n d i v e r g e n c e a m o n g t h e D. obscura g r o u p s p e c i e s is a s g r e a t a s t h e d i v e r g e n c e b e t w e e n

t h e s e s p e c i e s a n d D. melanogaster, a s p e c i e s o f a

d i f f e r e n t g r o u p .

B a s e d o n 6 v a l u e s ( T a b l e 4) , t w o p h y l o g e n e t i c t r e e s

r e l a t i n g t h e 16 d i f f e r e n t n u c l e o m o r p h s b e l o n g i n g t o

t h e obscura g r o u p s p e c i e s w e r e c o n s t r u c t e d (Fig . 3).

O n e (F ig . 3a ) b y t h e U P G M A m e t h o d ( S n e a t h a n d

S o k a l 1 9 7 3 ) a s s u m e s a c o n s t a n t r a t e o f e v o l u t i o n i n

t h e d i f f e r e n t l i n e a g e s . T h e o t h e r (Fig . 3 b ) u s e s t h e

n e i g h b o r - j o i n i n g m e t h o d ( S a i t o u a n d N e i 1 9 8 7 ) ,

w h i c h a l l o w s v a r y i n g r a t e s o f e v o l u t i o n . A l t h o u g h

t h e l a t t e r m e t h o d p r o d u c e s a n u n r o o t e d t r e e , a r o o t

w a s p l a c e d a t t h e m i d p o i n t o f t h e l o n g e s t p a t r i s t i c

i n t e r n u c l e o m o r p h d i s t a n c e ( F a r r i s 1 9 7 2 ) , i n o r d e r

t o f a c i l i t a t e t h e c o m p a r i s o n s o f t h e r e l a t i o n s h i p s

a m o n g s p e c i e s i n b o t h t r e e s . I t c a n b e s e e n t h a t t h e

t w o t r e e s g i v e t h e s a m e o v e r a l l p a t t e r n o f r e l a t i o n -

L

a

7 o " - - . - . . .L_

I - !

"[ I ' !

I

I

i i

! i " !

6 0 5 0 4 0 3 0 20 10 0 I I I I I ! J

127

D. 8 U b o b s c u r e - 3

D. s u b o b s c u r a - 8

D. s u b o b s c u r a - 1 3

D i s u b o b s c u r a - 2

D. m a d e i r e n s i s

D . g u a n c h e

D. s u b s i l v e s t r i s

D. obscura

D. ambigua - 3

D. a m b i g u a - 2

D. a r n b i g u a - 1

D b i fasc ia t a

D . t r i s t i s

D.pseudoobs c u r a - 3

D .pseudoobscu r a - 2

D . p s e u d o o b s c u r a - 1

D x 1 0 0 0

I

D. pseudoobscura - 1

D. pseudoobscura -3

D. pseudoobscur a- 2

D s u b o b s c u r a - 2

D. s u b o b s c u r a - 8

D . s u b o b s c u r a - 1 3

D . s u b o b s c u r a - 3

D.m adei rens i s

D . g u a n c h e

D. subsi Ivest ri s

D. ob 8cura

D. ambigua - 2

D. amb lgua- 3

D. arnbigua - 1

D. b l f a s c l a t a

D . l r i s t l s

b 10 ! ! D x 1 0 0 0

Fig, 3. Phylogenetic relat ionships among the 16 different nuc leomorphs representing the nine Drosophila obscura subgroup species, based on the 8 values given in Table 4. a U P G M A method and b neighbor-joining method, with branches drawn to scale.

ships, particularly when the standard errors depicted on the branching points of the U P G M A tree, as proposed by Nei et al. (1985), are taken into account.

Discussion

l~traspecifzc mtDNA Polymorphism

Two statistical measures are generally reported in studies ofintraspecific mtDNA polymorphism. One of them, nucleotide diversity, ~r, gives the average

mtDNA nucleotide diversity among individuals (Nei and Tajima 1981); the other, 6, is the nucleotide divergence between nucleomorphs (Nei and Tajima 1983). As the first one takes into account the pop- ulation frequency of each nucleomorph, we can only present ~r values for the species D. subobscura and D. melanogaster. For these species sufficient strains from the same locality, Tenerife island, have been analyzed with an identical set of endonucleases (Afonso et al. 1990 and unpublished results). Vari- ability per nucleotide in Tenerife for D. subobscura

128

(~r = 0.005) is five times higher than for D. mela- nogaster (Tr = 0.001). The overall variability in the Old World populations is also higher in D. subob- scura (~r = 0.008) (Rozas et al. 1990) than in D. melanogaster (Tr = 0.002) (Hale and Singh 1987). This difference could be explained by different de- mographic histories between both species. The two nucleomorphs presented in this study as represen- tatives of D. melanogaster (Table 2 and Fig. 2) are due to different patterns found with endonucleases HaeIII and HindIII. Our patterns A and B corre- spond to the same patterns of Shah and Langley (1979), and have a frequency in Tenerife of 0.85 and 0.07 respectively. Therefore, according to Hale and Singh (1987) the Tenerife population ofD. mel- anogaster is a typical Old World population.

For the rest of the species insufficient strains were assayed to determine ~r values. Nevertheless it is notable that no polymorphism was detected in D. guanche, an endemism for which 13 strains were analyzed. There are no obvious differences in the intra nor in the interspecific mean ~ values among the D. obscura subgroup species, D. melanogaster presents a lower intraspecific mean ~ value than the obscura subgroup species (Table 3).

Intraspecific polymorphism has previously been studied for D. pseudoobscura. Hale and Beckenbach (1985) found that HpalI restriction sites were hy- pervariable in this species (nine different patterns for 33 strains analyzed). The same is true in this study in which three different HpalI patterns were found from eight strains analyzed. Patterns A and B (Fig. 2) correspond to A and E of Hale and Beck- enbach (1985), and our C was not previously de- tected by these authors. This fact confirms the site hypervariability for this enzyme in D. pseudoob- scura.

Phylogenetic Relationships among Related Species

Mitochondrial DNA restriction site maps have often resolved phylogenies of related taxa where other ap- proaches have failed (Wilson et al. 1985; Avise 1986; Moritz et al. 1987). This is the case in the present study for the relationship of the related Palearctic species D. subobscura, D. guanche, and D. madei- rensis. By chromosomal homology, K.rimbas and Loukas (1984) suggested a first dichotomy giving D. guanche and a later one in which D. rnadeirensis and D. subobscura split off [but see also Molt6 et al. (1987) and Papaceit and Prevosti (1989) who report new differences between D. guanche and D. sub- obscura and D. madeirensis and D. subobscura, re- spectively]. Electrophoretic data are more abun- dant, though less conclusive. In a first study Cabrera et al. (1983) found the following order of relation-

ships: D. guanche-D, madeirensis < D. subobscura- D. madeirensis < D. subobscura-D, guanche. Nevertheless, these authors emphasized that, in spite of the greater morphological similarity of the two insular endemisms when compared with D. sub- obscura, the genetic distance between them was only slightly less than the distance of both from D. sub- obscura. In contrast, Loukas et al. (1984) have sug- gested a closer relation of D. madeirensis with D. subobscura, which are both more distant from D. guanche. Larruga and Pinsker (1984) reached the same conclusion based on the genetic divergence of chromosome O in these species. Molecular studies on highly repetitive DNA have also shown that D. guanche has a species-specific subunit that is not present in D. madeirensis and D. subobscura, indi- cating a more distant position ofD. guanche (Bach- mann et aI. 1989). Recently, Cariou et al. (1988) proposed a new relative pairing of these species. Drosophila madeirensis-D, guanche would be the closest pair as in Cabrera et al. (1983) but differing from these authors in the fact that the distance be- tween D. subobscura-D, guancheis smaller than that ofD. subobscura-D, madeirensis. Cariou et al. (1988) suggested that these discrepancies might be indic- ative of ambiguity regarding their relative genetic divergence. This is clearly not the case at the mtDNA level. It can be seen in Table 4, that the divergence between D. subobscura and D. madeirensis is un- mistakably less than that between D. guanche and D. madeirensis, and this pair, in turn, is more closely related than the D. guanche-D, subobscura one. These facts are graphically reflected in the trees of Fig. 3, where the standard errors on the UPGMA tree branch points confirm that the relationships found are statistically relevant. I f we assume that nucleotide divergence values in these trees are pro- portional to divergence times, distances between species can be converted to time applying the equa- tion, t [millions of years (Myr)] = 1/k (%) x 0.5 x

(%), which assumes that the rate of evolution of mtDNA in Drosophila, k, is 1.7%/Myr (Caccone et al. 1988). Divergence between D. guanche and the ancestor that gave rise to D. madeirensis and D. subobscura would have occurred around 3 Myr ago, whereas D. madeirensis and D. subobscura split off 1 or 2 Myr ago.

Another related cluster of Palearctic species de- tected with our mtDNA data is that formed by the species D. ambigua, D. obscura, and D. subsilvestris (Fig. 3). This relationship was reported at the mor- phological level by MacIntyre and Collier (1)86), using the data of Buzzati-Traverso and Scossiroli (1955), and has been repeatedly confirmed with elec- trophoretic data (Lakovaara et al. 1976; Pinsker and Buruga 1982; Loukas et al. 1984; Cariou et al. 1988). Nevertheless, there is an important difference be-

tween the morphological and electrophoretic clus- ters compared with the mtDNA one. In the former cases, D. tristis always appears as closely related to D. obscura, but with mtDNA data they are far apart (Fig. 3). Though length variants could confound re- striction site comparisons (Moritz et al. 1987), it seems not to be the cause in this case, because length variation among these species only affects the A+ T- rich region: conserved sites for HindIII and SacI are coincident in all the species. On the other hand, mtDNA phylogenies of closely related species are SOmetimes different from nuclear marker-derived trees (Solignac et al. 1986a; Caccone et al. 1988). Frequently, these differences are caused by in- trogression and/or stochastic lineage sorting, phe- nomena for which the mtDNA is more sensitive (Moritz et al. 1987).

Phylogenetic Relationships among Distant Species

The resultant picture for the more distantly related Species of the obscura subgroup shows five differ- entiated clusters at the mtDNA level. One Nearctic, represented by D. pseudoobscura, and four Palearc- tic, the two above-mentioned formed by D. sub- obscura, D. madeirensis, D. guanche, and D. am- bigua, D. obscura, D. subsilvestris, and two more represented each by one species, D. bifasciata and D. tristis (Fig. 3). Taking into account the standard errors on the tree nodes, the different Palearctic clusters are as distant between themselves as to the Nearctic species D. pseudoobscura. This result seems to favor the hypothesis that a common ancestor gave r~se to various lines in the Palearctic, one of which gave rise to the American species and the others to the different groups of European species (Cabrera et al. 1983). This conclusion must be treated with cau- tioa because of the discrimination limits of our mtl:)NA data.

The upper limit within which mtDNA restriction site maps are useful for phylogenetic analysis is bOUnded by substitutional saturation at rapidly evolving sites, which arises at about 15% overall Sequence divergence (Brown et al. 1979; Avise 1986; Me, ritz et al. 1987). In our study the resolution pow- er seems to be rather less. Table 3 gives the mean distance values of each species with all the other ob~cura subgroup species. It can be seen that the Value of D. melanogaster, an outgroup species, is not larger than the values of species within the ob- S.CUra group. Moreover, when a tree was constructed including D. melanogaster, this species clustered Within the group ofD. ambigua, D. obscura, and D. SUbsilvestris. For this reason this species was not inCluded as an outgroup species in the construction of trees. A similar fact was found by Caccone et al.

129

(1988), where an outgroup species, Drosophila taka- hashii, clustered within species of the D. melano- gaster subgroup. Due to these phylogenetic incon- sistencies, we believe that the mean divergence of D. melanogaster with all the other species (12 +_ 2%) corresponds to the plateau of the curve of mtDNA sequence divergence detected with the restriction site mapping method using our set of enzymes. We attribute this difference to the fact that in our study nucleotide substitutions were detected by the pres- ence or absence of restriction sites found with en- donucleases that mainly recognized 6-bp sequences. In the initial rapid phase of divergence, nucleotide substitutions can be equated with changes in restric- tion sites, but with increased divergence times, more nucleotide substitutions can accumulate in a 6-bp sequence than in a 4-bp sequence, and the real num- ber of substitutions can only be determined by DNA sequence analysis.

Differences in Size

Mitochondrial DNA length variation in Drosophila seems to arise from differences in copy-number of tandemly repeated sequences in the A+T-rich re- gion (Fauron and Wolstenholme 1976, 1980). In the D. melanogaster species subgroup, mitochondrial genome size variation fits in well with phylogenies based on the cleavage maps of mtDNA (Solignac et al. 1986b). This seems to be the case for the D. obscura subgroup species of this study. Well-related species have mtDNA genomes with similar sizes, whereas the five distant clusters identified differ in the lengths of their mtDNA (Table 3).

Rates of mtDNA Evolution

Though a constant rate of mtDNA evolution has to be assumed in order to estimate divergence times between well-differentiated taxa, there is increasing evidence that mtDNA evolutionary rates can vary between closely related lineages, possibly as a result of demographic influences on the rate of fixation of transient potymorphisms (reviewed in Moritz et al. 1987). Our experimental data confirm this hypoth- esis: Both trees, constructed by UPGMA, where constant rates are assumed (Fig. 3a), and by the neighbor-joining method, which does not assume constant rates (Fig. 3b), give the same overall pat- tern of relationships. Furthermore, the number of nucleotide substitutions of each nucleomorph to the root of the phylogenetic tree constructed by the sec- ond method is rather similar in all of them as shown by the nonsignificant chi-square obtained (X 2 = 12.5, df = 15) and by the small standard error of the overall mean (63.2 ___ 1.8) • 103 (Fig. 3b). These facts confirm a constant rate of mtDNA evolution

130

b e t w e e n s p e c i e s . H o w e v e r , 2 3 % o f t h e t o t a l v a r i a n c e

o f t h i s m e a n is d u e t o i n t r a s p e c i f i c v a r i a n c e , i .e . ,

d i f f e r e n t e v o l u t i o n r a t e s b e t w e e n n u c l e o m o r p h s

w i t h i n s p e c i e s .

Acknowledgments. We thank Dr. A. Prevosti and Dr. B. Saka- guchi for providing flies, Dr. D.R. Wolstenholme and Dr. R. Garesse for providing us with their mtDNA clones olD. yakuba and D. melanogaster, respectively, and Dr. M. Nei for sending us the program for calculating delta values and the data for the neighbor-joining method. We also thank Dr. A.J. Jeffreys for his commentaries and stylistic corrections on the manuscript. This research was supported by grant PR83-0396 from CAICYT to V.M. Cabrera.

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Received October 25, 1989/Revised February 26, 1990