MITOCHONDRIAL DNA EVOLUTION IN

Copyright 0 1983 by the Genetics Society of America

MITOCHONDRIAL DNA EVOLUTION IN MICE

STEPHEN D. FERRIS,* RICHARD D. SAGE,**t ELLEN M. PRAGER,* UZ1 RITTE**' and ALLAN C. WILSON*

*Department of Biocheinistry and +Museum of Vertebrate Zoology, University of Calijirnia, Berkeley, Californiu 94720

Manuscript received August 4, 1981 Revised copy accepted July 21, 1983

ABSTRACT

This study extends knowledge of mitochondrial DNA (mtDNA) diversity in mice to include 208 animals belonging to eight species in the subgenus Mus. Highly purified mtDNA from each has been subjected to high-resolution restriction mapping with respect to the known sequence of one mouse mtDNA. Variation attributed to base substitutions was encountered at about 200 of the 300 cleavage sites examined, and a length mutation was located in or near the displacement loop. The variability of different functional regions in this genome was as follows, from least to most: ribosomal RNA, transfer RNA, known proteins, displacement loop and unidentified reading frames.-Phylogenetic analysis confirmed the utility of the SAGE and MARSHALL revision of mouse classification, according to which there are at least four species of commensal mice and three species of aboriginal mice in the complex that was formerly considered to be one species. The most thoroughly studied of these species is Mus domesticus, the house mouse of Western Europe and the Mediterranean region, which is the mitochondrial source of all 50 of the laboratory strains examined and of the representatives of wild house mice introduced by Euro- peans to North and South America during the past few hundred years.- The level of mtDNA variation among wild representatives of M . domesticus is similar to that for the Eastern European house mouse (M. tnusculus) and several other mammalian species. By contrast, among the many laboratory strains that are known or suspected to stem from the pet mouse trade, there is little interstrain variation, most strains having the 'old inbred" type of domesticus mtDNA, whose frequency in the 145 wild mice examined is low, about 0.04. Also notable is the apparent homogeneity of mtDNA in domesticus races that have fixed six or mare fused chromosomes and the close relationship of some of these mtDNAs to those of karyotypically normal mice.-In addition, this paper discusses fossil and other evidence for the view that in mice, as in many other mammals, the average rate of point mutational divergence in mtDNA is 2-4% per million years. From this, it is estimated that the commensal association between mice and our ancestors began more than a million years ago, i.e., at an early stage in the evolution of Homo erectus.

0 make efficient use of the genetic variation present in wild mice, genet- T icists need a realistic picture of how that variation is organized with respect to populations and species of wild mice. Accurate information about the extent of genetic divergence within and between such populations in dif-

' Permanent address: Department of Genetics, Hebrew University, Jerusalem 91904, Israel

Genetics 105 681-721 November, 1983.

682 FERRIS ET AL.

ferent parts of the world is also a prerequisite for investigation of the genetic basis of evolutionary change. House mice and related species may prove to be one of the best groups with which to examine the genetic basis of evolutionary change because they are being studied more intensively at many levels of biological organization than any other group of vertebrates.

Until recently there was no quantitative phylogenetic framework with which to organize knowledge of the genetic and phenotypic diversity among the lineages leading to house mice and their relatives. SAGE’S (1 98 la) comparative electrophoretic study of proteins encoded by 56 loci was a major step toward remedying this lack. His findings, supplemented by those of THALER, BON- HOMME and BRITTON-DAVIDIAN (1981) and BONHOMME et al. (1983), and considered together with MARSHALL’S morphological comparisons ( MARSHALL 198 1 ; MARSHALL and SAGE 198 l), showed that the traditional classification of house mice and their relatives (SCHWARZ and SCHWARZ 1943) reflected neither the genetic relationships nor the morphological similarities among these mice. They, therefore, revised the classification to emphasize the major genetic groups of house mice and their relatives. The present study deals with the following species recognized by SAGE and MARSHALL:

I. House mice and their close relatives A. Commensal mice

1. M. domesticus, Western Europe, North Africa, Near East (including

2. M. musculus, Eastern Europe 3. M. molossinus, Northern Asia 4. M. castaneus, Southern Asia

5. M. spretus, Western Europe, North Africa 6. M. abbotti, Eastern Europe 7. M. hortulanus, Eastern Europe

8. M. cemicolor, Southeast Asia

M. poschiavinus, the tobacco mouse)

B. Aboriginal mice

11. More distantly related species

An important purpose of the present paper is to test the utility of this nuclear genetic classification by detailed restriction analysis of mitochondrial DNA from mice representing all of these species. Previous mtDNA comparisons have provided valuable evidence regarding the relatedness of certain mouse lineages (YONEKAWA et al. 1980, 198 1, 1982; KING, SHADE and LANSMAN 198 1 ; FERRIS, SAGE and WILSON 1982; FERRIS et al. 1983a.b). These studies involved either a small number of lineages or used few restriction endonucleases. The present paper reports on genetic variation at approximately 300 mapped restriction sites in the mtDNAs of 208 mice. In addition to giving information that reinforces the SAGE and MARSHALL classification, particularly with regard to the reality and distinctiveness of M. domesticus, this mtDNA investigation throws additional light on the geography of mtDNA variation in wild mice, the origin of numerous inbred strains and of chromosomal races and the susceptibility of different parts of the mitochondrial genome to base substitution. Furthermore,

MITOCHONDRIAL DNA EVOLUTION IN MICE 683

a new perspective emerges regarding the time at which the commensal association between mice and protohumans began.

MATERIALS AND METHODS

Mice: Table 1 lists the 50 "laboratory" strains of mice examined and their sources. Mice trapped in the wild and laboratory descendants of separate lines of wild-caught females provided an additional 158 animals from 51 localities, as listed in Table 2.

Preparation, digestion and electrophoresis of mitochondrial DNA: MtDNA was purified from soft tissues of single mice in all but a few instances, where two to three littermates were pooled. The mtDNA was highly purified by ultracentrifugation according to BROWN, GEORGE and WILSON (1979), as previously described (FERRIS, SAGE and WILSON 1982; FERRIS et al. 1983a,b). About 10 ng of mtDNA was digested with each restriction endonuclease (New England Biolabs). Fragments were end-labeled with '*P and electrophoresed in 1.2% agarose or 3.5% polyacrylamide gels, which were autoradiographed for 3-24 hr at -80" (BROWN 1980). Estimates of fragment sizes were made by comparing them with the known fragment sizes of the mouse mtDNA of known sequence (BIBB et al. 1981). The phage DNAs PM2 cut with HindII1, X cut with EcoRI and 6x174 cut with HincII provided additional size standards.

The mtDNAs of mice 1-91 (Tables 1 and 2) were digested with 11 different restriction enzymes, recognizing six bases (HindIlI, XbaI, HincIl, AccI), five bases (AvaII) and four bases (FnuD11, HpaI1, HaeIII, TaqI, MboI, HinjI). The remaining samples (save M. abbotti and M. hortulanus) were digested with MboI and Hinfl, which are the most discriminating (FERRIS et al. 1983a). In addition, old inbred M. domesticus and M. spretus mtDNA were subjected to single and double digestion with 12 six-base enzymes to produce the cleavage map for M. spretus. These 12 enzymes were designated with a single-letter code as follows: EcoRI, b HindIII, HPaI, BgllI, e XbaI, f BamH1, g PstI, Aval, y BglI.

Fragment patterns and cleavage maps: Each fragment pattern produced by a given enzyme was assigned a capital letter, with A being reserved for the pattern appearing in the mtDNA of known sequence (BIBB et al. 1981). We continue the practice introduced by FERRIS, SAGE and WILSON (1982) and FERRIS et al.. (1983a,b) which designates the patterns chronologically as they are discovered, so that alphabetical proximity to A does not necessarily correlate with degree of similarity to the A pattern.

Cleavage maps for each enzyme were constructed by the sequence comparison method (CANN, BROWN and WILSON 1982; CANN and WILSON 1983; FERRIS et al. 1983a,b), in which one maps the sites needed to account for each fragment pattern by comparison with the known sequence (BIBB et al. 1981). It was not possible to derive complete maps for all enzymes for the non- domesticus types of mtDNA by this method, and, in such instances, fragment sizes are reported ($ APPENDIX). In the case of M. spretus, mtDNA was also mapped by the double-digestion method.

Evolutionary trees were constructed by the parsimony method (FERRIS, WILSON and BROWN 1981; FERRIS et al. 1983a,b), which does not assume a constant rate of mtDNA evolution. Restric- tion enzyme sites were used as characters for tree construction with domesticus types of mtDNA or with the musculus and molossinus types. Fragment sizes were used as characters to investigate the branching order of the more distant lineages to one another.

Two approaches were used to estimate the extent of point mutational divergence between pairs of mtDNAs, as described in detail previously (FERRIS et al. 1983b): the site method is based on map comparisons and employs equation 16 of NEI and LI (1979), whereas the fragment method is based on the fraction of shared fragments and employs equation 20 of NEI and LI (1979). The site method, which underestimates actual sequence divergence to a lesser extent, was employed whenever complete or nearly complete maps could be derived.* The fragment method supplemented the site method for comparison of the more distant taxa. In all cases the final value was obtained by weighting according to the number of six-, five- and four-base sites compared.

PvuI1, i SacI, 1 XhoI,

* For mtDNAs among mice 81-86 (Table 2). it was possible to infer the number of site changes from one another, even when the site locations could not be determined exactly.

TABLE 1

Sources of laboratory mouse strains

Oldest nonlaboratory Oldest nonlaboratory Mouse no. and strain Mouse no. and strain source, date and category source, date and category

IM. domesticus 1 DBA/2N

2 BALB/cJ 3 C3H 4 AU/SsJ 5 C57BL/6J

6 C58/J 7 SWR/J

9 AKR/Cum

10 DBA/LiA 11 129/MA 12 BALB/cByA 13 BALB/cCdA 14 A/BrAf 15 A2G/GA 16 C3H/HeAf 17 CBA/BrA 18 C57BL/LiA 19 C57BLlImrHeA 20 BIMA/A 21 RIIIS/J

22 FVB/NA 23 SJL/JA 24 STS/A

25 GRS/A 26 MAS/A

Dealer, Massachusetts,

Dealer, Ohio, 1913, A As for no. 2, A (?) As for no. 2, A Dealer, Massachusetts,

As for no. 5, A Dealer, France, <1920,

Dealer, New Jersey,

Dealer, Pennsylvania,

As for no. 1, A (?) As for no. 1, A As for no. 2, A As for no. 2, A (?) As for no. 2, A (?) As for no. 2, A As for no. 2, A As for no. 2, A As for no. 5, A As for no. 5, A (?) As for no. 2, A Unknown, France,

As for no. 7, B As for no. 7, B Unknown, Switzerland,

-1930, B As for no. 24, B As for no. 24, B

1909, A

1921, A

A

1922, A

1928, A

1928, B

27 LIS/A 28 LTS/A 29 TSI/A

30 DD/HeAf

31 WLL/BrAf

32 020/A

33 CE/JA 34 NMRI/Lac 35 AKR/FuRdA 36 NMRI/Navy 37 SL/NiA

38 NZB/Ibm

39 NZW/Ola 40 MOR/Cv 41 PAC/Cv

42 IS/Cam/J 43 SF/Cam/J

44 SK/Cam/JEi 45 NZB/BlNJ 46 NZB/Bom 47 NZO/Dus 48 NMRI/Bom iM. nolossinus” 87 KL/oci 88 MOLS/JA

As for no. 24, B As for no. 24, B Unknown, Germany,

Unknown, Germany,

Dealer, Norway, 1929,

Dealer, Netherlands,

Wild, Illinois, 1920, B As for no. 7, B As for no. 9, A As for no. 7, B Unknown, Switzerland,

-1930, B Unknown, England,

1930, B (?) As for no. 38, B Wild, Ohio, -1963, C Wild, Pennsylvania,

Wild, Israel, 196 1, C Wild, California, 1951,

-1930, B

C1920, B

B

1931, B

1967, C

Wild, Wales, 1962, C As for no. 38, B As for no. 38, B As for no. 38, B As for no. 7, B

Wild, Japan, 1967, C As for no. 87, C

Genealogical information including place and time of origin is based on LYNCH (1969), BIEL- SCHOWSKY and GOODALL (1970), CONNOR (1978), MORSE (1978a,b), POTTER and KLEIN (1979), STAATS (1964, 1980), RICE and O’BRIEN (1980), SAGE (1981b), HILKENS et al. (1981), FERRIS, SAGE and WILSON (1982), FERRIS et al. (1983a), and references therein, and on personal commu- nications from J. HILCERS and V. CHAPMAN. Strains in category A belong to the old inbred group considered in FERRIS, SAGE and WILSON (1982); strains in category B have been in the laboratory for at least 50 years, and many probably have a genealogical connection to A; strains in category C are more recently derived from known wild localities (6 [email protected] of laboratory mzce- for further details). Information is given for the matrilineal source in cases in which females and males from different sources were mated. We obtained mice from: National Cancer Institute, Bethesda, Maryland (no. 1); The Jackson Laboratory, Bar Harbor, Maine (nos. 2, 4-8, 21, 42-45); M. LIEBERMAN, Stanford University, Stanford, California (no. 9); J. HILGERS and R. NIKKELS, Netherlands Cancer Institute, Amsterdam (nos. 10-20, 22-33, 35, 37, 88); K. FISCHER LINDAHL, Basel Institute for Immunology, Basel, Switzerland-see also FERRIS et al. (1983a) and FISCHER LINDAHL and HAUSMANN (1983) (nos. 34, 36, 38, 39, 46-48); V. CHAPMAN, Roswell Park Me- morial Institute, Buffalo, New York (nos. 40, 41, 87). MtDNA from the LA9 cell line, whose sequence was determined (BIBB et al. 1981), represented C3H (no. 3) and was a gift from W. M. BROWN. Strains 1-9 are the old inbreds examined by FERRIS, SAGE and WILSON (1982); strains 21, 34, 36 and 38-48 were also previously studied (FERRIS, SAGE and WILSON 1982; FERRIS et al. 1983a). Strains 48 and 87 are not inbred. Strain 38 was previously designated NZB/Fiill (FERRIS et al. 1983a).

“See Table 2, mouse 86. 684


TABLE 2

Collecting localities for wild-caught or laboratoryraised descendants of wild mice subjected to mitochondrial DNA analysis

Mouse numbef Place and time of collection from the field

- 49*, 92*-97*

- 50*, 98*-102* 51* - 52*, 103*-110* -

- 53*, E* 111*-122* 123*, 124* 55*-57* 58 125* - 59-64

- 65, 126, 127 - 66, 128-134

- 67, 135-139 68, 140 - 69, 141, 142 - 7 0 , z , 143, 144 72

-

- 73 - - 7 4 , E , 145, 146 - 76, 147-149 150*-153* 154*, 155* 156*-158* 159*-162* 163*-167* 168*-171* 172* 173*, 174* 175* 176* 177**-179* 180*

77 181 -

81 -

M. domesticus California, Santa Barbara Co., BUELLTON 1980 (#49), 1981, by R. D.

California, Napa Co., NAPA, 1979 (#50), 1981, by R. E. JONES California, Contra Costa Co., ORINDA, 1979, by 0. P. PEARSON California, Sonoma Co., PETALUMA, 1980 (#52, 103), 1981, by R. D.

California, Alameda Co., SAN PABLO, 1979, 1980, by J. HAFNER California, Contra Costa Co., ANTIOCH, 1981, by P. BRYLSKI California, Alameda Co., BERKELEY, 1982, by U. RITTE INDIANA, Vanderburgh Co., Evansville, 1981, by M. FERRIS MARYLAND, Queen Anne Co., Centreville, 1978, by M. POTTER ILLINOIS, Chicago, 1981, by R. D. SAGE PERU, Lima Dpto., 0.5 km from Nafia, 1976. Designated Peru-Coppock,

Israel, JERUSALEM, 1979, by U. RITE Egypt, FAIYUM Governate, El Faiyum Depression, 1979, by H. Hoocs-

Egypt, GIZA Governate, near Bashtil, 1979, by H. H ~ S T R A A L Morocco, Tafilalt Oasis, ERFOUD, 1978, by R. D. SAGE Morocco, 7 km NW AZROU, 1978, by R. D. SAGE Yugoslavia, Dalmatia, ZADAR Is.,-1980 or 1981, by A. GROPP Yugoslavia, Dalmatia, METKOVIC, 1979, by R. D. SAGE Switzerland, Vaud Canton, Givrins, near NYON, 1978, by R. D. SAGE Italy, Binasco SW MILAN, 1982, by A. GROPP. Designated Mil 11 Italy, CITTADUCALE near Abruzzi, 1979, by A. GROPP. Designated CD England, border Norfolk and Suffolk Co., BUNWELL, 1980, by R. J. BERRY England, TAUNTON, Chedzoy near Bridgwater, 1982, by R. J. BERRY Scotland, Orkney Is., N. WESTRAY, 1981, by R. J. BERRY Scotland, Orkney Is., S. WESTRAY, 1981, by R. J. BERRY Scotland, Orkney Is., S. RONALDSAY, 1981, by R. J. BERRY Scotland, Orkney Is., STENNESS, 1981, by R. J. BERRY Scotland, Shetland Is., OLLABERRY, 1981, by R. J. BERRY Scotland, Shetland Is., YELL, W. Sandwick, 1981, by R. J. BERRY Scotland, Shetland Is., Wester QUARFF, 1981, by R. J. BERRY Denmark, S. Jutland, HADERSLEV, 1982, by J. T. NIEUEN West Germany, LUBECK (Eichholz), 1982, by A. GROPP West Germany, MARGARETHENHOF near Segeberg, 1982, by A. GROPP

M. domesticus, formerly M. poschiavinus' Switzerland, Grisons Canton, Zalende, POSCHIAVO, 1978, by R. D. SAGE Italy, Sondrio Prov., TIRANO, 1979, by R. D. SAGE

Denmark, HOV, 1981, 1982, by J. T. NIELSEN Denmark, SKIVE, 1974, by J. T. NIELSEN; maintained by V. CHAPMAN Denmark, VIBORG, 1974, by J. T. NIELSEN; maintained by V. CHAPMAN Sweden, MALMO, 1982, by J. T. NIEUEN Czechoslovakia, Moravia, Studenec, approx. 35 km W. BRNO, 1978, by B.

SAGE

SAGE

supplied by M. E. WALLACE

TRAAL

M. musculus

686 FERRIS ET AL.

TABLE 2-Contiizued

Mouse number‘ Place and time of collection from the field

- 82

- 83, fi 205*

Czechoslovakia, Slovakia, Slideckovce, approx. 50 km E. BRATISLAVA,

Yugoslavia, Serbia, BELGRADE, 1979, by D. KATARANOVSKI Poland, TUREW, 1982, by W. Z. LIDICKER

Japan, OKINAWA, Naha, 1980, by T. NAKADA Japan, KYUSHU, Fukuoka, 1967, by F. HAMAJIMA. Sent to M. POTTER,

from whom #86 comes. #87 and 88 (Table 1) were derived from this same stock and Potter colony

1978, by M. KMINIACK

M. rnolossiiius

- 85 - 86

M. castaiieus

- 89 Thailand, Thonburi Prov., THONBURI, ca. 1974, by J. T. MARSHALL; maintained by V. CHAPMAN.

M. spretus

M. remicolor popneus - 90

- 91

Spain, CADIZ prov., 8 km E. Puerto Real, 1977, by R. D. SAGE

Thailand, SARA BUR1 Prov., Phu Namtok, 21 km ENE. Sara Buri, before 1976, by J. T. MARSHALL; maintained by R. CALLAHAN

M . abbotfi 206 Yugoslavia, Macedonia, 6 km NW. GRADSKO, 1979, by R. D. SAGE

Austria, Burgenland, Kleylehof, 6 km ENE. HALBTURN, 1979, by R. D.

Yugoslavia, Serbia, Debeljaca, approx. 20 km N. PANCEVO, 1979, by D.

M. hor~uluiius 207

208 SAGE

KATARANOVSKI

MtDNA from samples which are underlined was analyzed with 11 restriction enzymes and the results presented in Table 3. MtDNA from the remaining samples (except nos. 206-208) was always analyzed with MboI and HiiifI (results in Table 4) and in some cases with several but not all of the other nine enzymes. An asterisk indicates that wild-caught animals were analyzed. (For several sequential numbers where all were wild-caught, the asterisk is appended to the first and last numbers.) Further details on the exact collecting localities are available from the authors.

“Samples 49-76, 78-84, 176, 177 and 182-205 were previously examined (FERRIS, SAGE and WILSON 1982; FERRIS et al. 1983a,b), in many cases with only a few restriction endonucleases.

*Sample 177 contributed the I pattern for FiiuDII (Table 7A, APPENDIX). ‘See DISCUSSION (Model for chroinosoinal Poolutiori). We join GROPP et al. (1982) in refraining from

assigning a separate taxonomic rank to chromosomal races within M . doinesticus, and we list M. poschiaviiius only for purposes of identification with MARSHALL and SAGE (1981) and MARSHALL (1981).

When the site methods were used for parsimony tree construction and for estimates of sequence divergence, cognizance was taken of the fact that in seven instances a single base change was recognized by two restriction enzymes (6 APPENDIX Tables 5A, 6A and 8A-12A for further details). In each instance, the change was counted only once.

RESULTS

Fragment patterns and corresponding maps Table 3 lists the fragment patterns resulting from digestion of the mtDNAs

of 91 mice with 11 restriction enzymes. These patterns were considered in


TABLE 3

Fragment patterns for mouse intDNA digested with 1 1 restriction enzymes"

Hind Xba H i m Acc Ana FnuD Hpa Hae T;q M P H F f Mouse number and kind 111 I I I I I I II II I11

M . domesticus Laboratory inice

A A A A K A A C A A A A A A A A D A

1-34, 38-40 cf: Table 1 35b AKR/FuRdA 36 NMRI/Navy 37 SL/NiA 41 PAC/Cv 42 IS/Cam/J 43, 44 SF/Cam/J, SKI

Cam/JEi 45-48 NZB etc.+ Ta-

ble l

A A A A A A A A A A B A

A A A A A A A A A A A A A A Q * A A A A A A Q A T A A A A A B A A P * D F M

B A B A C A A E B* J N

B A A D C H B A A A A Wild mice A A A 49 California-Buellton

50, 5 1 California-Nap,

52 California-Petaluma 53, 54 California-San

55-57 Indiana 58 Maryland 59-64 Peru 65 Israel-Jerusalem 66 Egypt-Faiyum 67 Egypt-Giza 68 Morocco-Erfoud 69 Morocco-Azrou 70, 71 Yugoslavia-Zadar 72 Yugoslavia-Metkovif 73 Switzerland-Nyon 74, 75 Italy-Mil I1

77 Switzerland-Pos-

Orinda

Pablo

76 Italy-CD

chiavo

A A A A A A A A

B A B A

A A A A A A

A A A B A C B A A D C H

A A B A A A B A B A B A B A B A B A B A B A B A B A B A

A A B A A A A A C

B C C A A C A A C A A A B C D G A A B C C C A A B A C B C D

B J I

A B * A A A B B A A D C H A A A A B A A A M B R C A A B B G J A A A C C D A A A D D E A C A E E F A A * O * G G J A A A B A C A D D B H K A A C D F I A A N B Y V A A B B I L

B A A E A M . musculus B C D

A A F D U C '

78 Denmark-Hov 79, 80 Denmark-Skive,

8 1 Czechoslovakia-Brno 82 Czechoslovakia-Bra-

83, 84 Yugoslavia-Bel-

Viborg'

tislava

grade

B A H A B B I L

B B B C

B B D A A D

A A B B I L D G G J L P

D F H K L Q

D E H L M R

B D A F D

B D A A E M . molossinus

A A E A A A

D G I M A ' P A A A A A A

85, 86 Japan 87, 88 KL/oci, MOLJ/JA

B D A A

6aa FERRIS E T AL.

TABLE 3-Continued

Hind Xbo Hinr Arc Aua FnuD Hpa Hue Taq Mbo Hinf Mouse number and kind 111 I I I I I1 I1 I 1 I11 I I I

M . custuneus 89 Thailand-Thonburi B F D G F E H J N B’ D’

90 Spain M. spretus

C E E H G F I K O P S IM. cerurrolor

9 1 Thailand-Sara Buri D G F I H G J L P Q E’

“An asterisk indicates changes from patterns previously reported (FERRIS, SAGE and WILSON 1982; FERRIS et al. 1983a). Corrections in the HpaII patterns were necessitated by discovery of the apparent reversal of two samples on a gel (nos. 53 and 69) and subsequent retesting. Those involving HaeIII patterns P and Q (compare FERRIS et al. 1983a) resulted from careful study of the complete maps for all enzymes for all taxa and the discovery that gain of an AccI site at 15924 must be accompanied by loss of the HaeIII site at 15923 (Tables 5A and 9A of APPENDIX); the changes in the HaeIII pattern are difficult to detect, as the band losses and gain occur in regions of multiple superimposed or closely spaced bands. HaeIII pattern 0 represents an additional change beyond pattern B (Table 9A). The TaqI change results from the recognition after map analysis that the pattern previously designated H is apparently the same as B. Patterns D, N, P for XbaI, MboI, and Hznfr, respectively, for M. musculus no. 205 are reported in FERRIS et al. (198313) and the APPENDIX. M. abbottr and IM. hortulanus (6 Table 2) were both digested with XbaI and AvaII, and each species yielded patterns unshared by any other taxon. M. hortulanus was also digested with HzncII, HpaII, HaeIII, Tag1 and Hznfr, and here also the patterns were unique to this species.

bA second animal was examined, with the same result as shown here for the enzyme AccI. ‘The patterns reported for Viborg mtDNA in FERRIS, SAGE and WILSON (1982) were in error

due to a mislabeled mouse; those in FERRIS et al. (198313) are correct.

relation to the known base sequence for one of these mtDNAs, and it was possible to infer maps for an average of 16’7 cleavage sites in each mtDNA. The APPENDIX gives the exact locations for many of these sites for each of the 32 types of mtDNA encountered in this survey. Table 3 also summarizes the results for mice 205-208 (footnote a).

MtDNAs from 1 13 more mice bearing domesticus mtDNA were digested with two restriction enzymes (MboI and Hinfr), and the resulting fragment patterns are listed in Table 4. The locations of the cleavage sites required to account for the 13 additional types of mtDNA revealed in this less complete restriction survey appear in the APPENDIX.

Domesticus MtDNA Genomic distribution of uariable and constant sites: The thin vertical lines in

Figure 1 give the approximate positions of the 206 cleavage sites mapped in domesticus mtDNAs. Of these, 68 are variable, i.e., present in some mtDNAs and absent in others, whereas 138 are constant, i.e., present in all domesticus mtDNAs tested. Both sorts of sites are distributed widely in the genome (Fig- ure 1).

Upon dividing the number of variable sites by the total number of sites in a region, one can estimate the variability in that region. This has been done for each mitochondrial gene, both in the bar graph (Figure 1) and in Table 5, from which it is evident that the order of variability, from least to most, is rRNA, tRNA, known proteins, displacement loop and unidentified reading frames.


TABLE 4

Fragment patterns produced by the restriction enzymes MboI and Hinfl in 147 mice bearing domesticus mtDiVA

Localities (and sample no.)

AA 6 A 0 1 AZ 4 sz 11 xz 2 AB 2 AY 3 AT 1 BA 1 AC 20

AU 4 zc 1 EF 2 FI 1 FM 1 VB' 1 UC' 2 cc 1 CD 1 CE 1 DE 1 T W 4 CH 18

RC 12 HK 1 IL 30

KL 3 IA' 1 GJ 6 JN 1 YV 4

BUELLTON, California (49, 92); BUNWELL, England (150-153) HADERSLEV, Denmark (1 76) BUELLTON (93, 94), BERKELEY (1 23, 124), California BUELLTON (95-97), NAPA (98-loo), ANTIOCH (1 11-1 15), California TAUNTON, England (154, 155) SAN PABLO, California (53, 54) YELL (173, 174), QUARFF (175), Shetland Is., Scotland SL/NiA (37) Centreville, MARYLAND (58) NAPA (50, 101, 102), ORINDA (51), California; ZADAR, Yugoslavia (70, 71, 143,

144); N. WESTRAY (156-158), S. RONALDSAY (163-167), STENNESS (168-171), Orkney Is., Scotland

S. WESTRAY, Orkney Is., Scotland (1 59-1 62) OLLABERRY, Shetland Is., Scotland (1 72) ERFOUD, Morocco (68, 140) NYON, Switzerland (73) ISRAEL (42) FAIYUM, Egypt (128) POSCHIAVO, Switzerland (77); TIRANO, Italy (181) GIZA, Egypt (135) FAIYUM, Egypt (66) GIZA, Egypt (136) GIZA, Egypt (67) FAIYUM (129). GIZA (137-139), Egypt PETALUMA, California (52, 103-1 10); INDIANA (55-57); ILLINOIS (125); FAI-

ANTIOCH, California (1 16-1 2 1); PERU (59-64) METKOVIk, Yugoslavia (72) CITTADUCALE, Italy (76, 147-149); HOV (78, 182-203), SKIVE (79), VIBORG

LUBECK, northern Germany (1 77-1 79) MARGARETHENHOF, northern Germany (180) JERUSALEM, Israel (65, 126, 127); AZROU, Morocco (69, 141, 142) ANTIOCH, California (1 22) MILAN, Italy (74, 75, 145, 146)

YUM, Egypt (130-134)

(80), Denmark; MALMO, Sweden (204)

This table lists all 31 types of domesticus mtDNA detected with the restriction enzymes MboI and H i n f l . The letters indicate the fragment patterns obtained with MboI and Hitzfl , respectively; this lettering system is consistent with that for these two enzymes in Table 3. Information about the locations of the cleavage sites responsible for these patterns appears in APPENDIX, Tables 11A and 12A. The 31 types are arranged in accordance with the evolutionary tree in Figure 3. Laboratory strains are included only when no wild representative of the same type was found in our present survey (AT and FM); FM has a documented wild origin (Table 1). (The remaining laboratory strains are of the AA, AB, CH and JN types-see Table 3.)

Gain mutations: Forty-three of the 68 variable sites are absent in the mouse mtDNA of known sequence and are assumed to be the result of base substitutions that have caused the gain of a site with respect to the known sequence. One can infer the exact location and nature of each of these gain mutations

690 FERRIS E T AL.

80 r

68 Variable Sites

138 Constant Sites

0 2 4 6 8 IO 12 14 16 Kilobases

FIGURE 1.-Locations of variable and constant cleavage sites in 38 types of doinesticvs mitochondrial DNA detected with two to 11 restriction endonucleases. The lines above the horizontal bar showing the mtDNA genes indicate the 68 variable sites and the lines below the bar the 138 constant sites. The mtDNA genome is oriented as in the BIBB et al. (1981) sequence and the genes are indicated as follows: ribosomal genes (12s and 16s); tRNA genes or spacers (black areas); genes encoding cytochrome oxidases I, I1 and I l l , ATPase, and cytochrome b (I, 11, 111, A, and b); unidentified reading frames presumed to encode additional proteins (1-6, A6L and 4L); displacement loop and adjacent noncoding regions (shaded portion). The histogram at the top of the diagram shows the degree of variability in each gene: the variable sites divided by the total sites and converted to percent. The histogram section with the asterisk indicates the variability for all 22 tRNAs collectively. Seven of the 68 variable sites are detected with two enzymes (see text and APPENDIX, Tables 5A, 6A and 8A-12A).

(see APPENDIX). Sixty-five percent of them are transitions, a value lower than that reported for sequenced regions of mtDNA from closely related primates (BROWN et al. 1982; GREENBERG, NEWBOLD and SUGINO 1983) and rats (BROWN and SIMPSON 1982) but in agreement with restriction data on human mtDNAs (CANN 1982).

Seventy-five percent of the 36 inferrable substitutions (including two losses- cf: APPENDIX, Tables 6A and SA) detected in protein-coding regions are silent (see APPENDIX), in agreement with the 78% observed by sequencing closely related primate mtDNAs (BROWN et al. 1982). Among the known proteins, the percentage of silent substitutions is 92, whereas the corresponding figure for the unidentified reading frames is 65.

Length mutations: One clear example of a length mutation was encountered in this comparison of domesticus mtDNAs. From the sizes of the restriction fragments produced by TaqI, HaeIII, MboI, HznfI and HpaII, we localized an addition of about 12 bp to the region from 15330 to 15736, which encom- passes 86 bp of two tRNA genes and 320 bp of noncoding DNA in the displacement loop. This addition was evident only in the tobacco mouse (am- ple 77). There could be additional minor variations in length among the domesticus mtDNAs examined; to detect such variation, however, a more thor-


TABLE 5

Variation in domesticus mitochondrial DNA genes

691

Number of sites Percent variability Inferred

Variable Constant mutations !!% Region (a) (b) (4 a + b a + b

Ribosomal RNAs 12s 16s Total

Transfer RNAs

Protein encoding Known proteins

CO I CO I1 ATP 6 CO I11 Cyto b Total

1 2 A6L 3 4L 4 5" 6" Total

Unknown proteins (URFs)

All proteins

Displacement loop

Total mitochondrial genome

1 10 3 16 4 26

3 15

4 19 1 10 2 4 7 7 4 12 18 52

8 10 4 5 0 0 5 2 0 0 4 8 8 11.5 3 2.5 32 39 50 91

3 7

60 139

1 3 4

3

4 1 2 7 5 19

8 7 0 5 0 4 13 4 41 60

6

73

9.1 15.8 13.3

16.7

17.4 9.1 33.3 50.0 25.0 25.7

44.4 44.4

71.4

33.3 41.0 54.5 45.1 35.5

30.0

30.2

9.1 15.8 13.3

16.7

17.4 9.1 33.3 50.0 31.3 27.1

44.4 77.8

71.4

33.3 66.7 72.7 57.7 42.6

60.0

36.7

The data are taken from the 199 sites detected among the 25 dom~sticus types of mtDNA digested with 1 1 enzymes and used to construct the tree in Figure 2 (below). The 73 inferred mutations derive from that tree. The 100a/(a + b) column is analogous to the histogram in Figure 1, whereas the lOOc/(a + b) column takes account also of multiple hits at a given site.

"A single TaqI site is part of both these overlapping genes.

ough examination of fragment mobilities would be required, like that con- ducted by CANN and WILSON (1983) for human mtDNAs.

Extent of diuergence and phylogenetic relationships: Table 6 summarizes the number of site differences for all possible pairs of the 25 types of domesticus mtDNA that were mapped for all 11 restriction enzymes. This number ranges from one to 21. From these values and the mean number of sites mapped per mtDNA, 166, we estimated the percent sequence divergence due to base substitutions. These divergences range from 0.05 to 1.55%, with an average of 0.77% (Table 6).

692 FERRIS ET AL.

TABLE 6

Mututiotirtl djffprmres ntnotig 25 t j p s of M. domesticus rr~itochondricil D,VA

Mice compared

1, etc. Inbreds" and 49 Cali-

35 AKR/FuRdA 36 NMRI/Navy 37 SL/NiA 58 Maryland 4 1 PAC/Cv-Pennsylvania 53, 54 California-San Pa-

50, 51 California-Napa,

70, 7 1 Yugoslavia-Zadar 45, etc. Inbreds' and 52, etc.

67 Egypt-Giza 66 Egypt-Faiyum 68 Morocco-Erfoud 77 Switzerland-Poschiavo 73 Switzerland-Nyon 42 IS/Cam/J-Israel 59-64 Peru 72 Yugoslavia-Metkovi; 74, 75 Italy-Mil I1 43, 44 SF/Cam/J, SK/Cam/

65 Israel-Jerusalem 69 Morocco-Azrou 76 Italy-CD 78 Denmark-Hov (mu) 79, 80 Denmark-Skive, Vi-

fornia

blo

Orinda

U.S.'

JEi

borg (mu)

1 35 36 37 58 41 53 50 70

- 1 1 1 1 1 3 3 4

0.05 - 2 2 2 2 4 4 5 0.06 0.11 - 2 2 2 4 4 5 0.07 0.12 0.14 - 2 2 4 4 5 0.07 0.12 0.14 0.15 - 2 4 4 5 0.07 0.13 0.14 0.15 0.15 - 2 4 5 0.21 0.26 0.27 0.29 0.29 0. i4 - 6 7

0.20 0.26 0.27 0.28 0.28 0.28 0.42 - 1

0.26 0.31 0.32 0.33 0.33 0.33 0.47 0.05 - 0.51 0.56 0.57 0.58 0.58 0.59 0.72 0.31 0.36

0.51 0.56 0.57 0.58 0.43 0.59 0.72 0.31 0.36 0.80 0.85 0.87 0.88 0.88 0.88 1.03 0.60 0.65 0.67 0.72 0.74 0.75 0.75 0.75 0.89 0.47 0.52 0.71 0.77 0.78 0.79 0.79 0.79 0.93 0.51 0.56 0.79 0.84 0.86 0.86 0.86 0.87 1.00 0.59 0.64 0.86 0.91 0.79 0.93 0.93 0.94 1.07 0.65 0.71 0.67 0.73 0.74 0.75 0.75 0.75 0.90 0.47 0.53 0.78 0.84 0.71 0.86 0.86 0.87 1.01 0.58 0.63 0.63 0.69 0.70 0.71 0.71 0.71 0.86 0.43 0.48 0.87 0.93 0.94 0.95 0.95 0.96 1.10 0.67 0.72

0.78 0.84 0.71 0.86 0.86 0.90 1.01 0.57 0.63 1.02 1.07 0.95 1.09 1.09 1.10 1.25 0.81 0.86 1.02 1.08 0.95 1.10 1.10 1.11 1.26 0.82 0.87 1.10 1.16 1.03 1.18 1.18 1.19 1.34 0.89 0.95 1.15 1.21 1.07 1.22 1.22 1.23 1.38 0.93 0.99

45

7

8 8 8 8 8

10

4

5 -

0.30 0.44 0.62 0.50 0.58 0.73 0.77 0.89 0.57 0.82

0.89 1.13 1.13 1.21 1.25

The upper right portion of the matrix gives the number of site differences determined from the cleavage maps (4 APPENDIX) constructed for the patterns in Table 3 for samples 1-80, 87 and 88. The lower left portion gives the percent nucleotide sequence divergence estimated with equation 16 of NEI and LI (1979). In contrast to Table 3, where the mice are listed according to species, lab strains and geography, they are here arranged according to degree of similarity, with

A tree depicting genealogical relationships among these 25 types of domesticus mtDNA appears in the upper part of Figure 2. This branching order allows the 25 types to be derived by divergence from a common ancestor with fewer mutations than any other branching order.

Thirteen additional domesticus types of mtDNA were detected and mapped with respect to the two most discriminating of the I 1 restriction enzymes, namely, MboI and Hinfl (see Table 4 and APPENDIX). Figure 3 depicts genealogical relationships among these 13 types (identified by solid circles) and the mtDNAs mapped for 11 enzymes. Although this tree was built only on the basis of the MboI and Hinfr data, it is nearly identical in topology with the tree based on 11 enzymes. The main exception is the EF lineage leading to a Morocco mouse (no. SS), whose genealogical position is slightly different in


67 66 68 77 73 42 59 72 74 43 65 69 76 78 79

7 11 9 10 11 12 10 11 9 12 I 1 14 14 15 16

8 I2 10 I 1 12 13 11 12 10 13 12 15 15 16 17 8 12 10 I1 12 11 11 10 10 13 10 13 13 14 15 8 12 10 11 12 13 11 12 10 13 12 15 15 16 17 6 12 10 11 12 13 11 12 10 13 12 15 15 16 17 8 12 10 11 12 13 11 12 10 13 12 15 15 16 17

10 14 12 13 14 15 13 14 12 15 14 17 17 18 19

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

5 9 7 8 9 1 0 8 9 7 1 0 9 1 2 1 2 1 3 1 4 4 6 8 7 8 10 11 12 8 11 12 15 15 16 17

- 8 8 7 8 10 11 12 8 11 12 15 15 16 17 0.60 - 12 11 12 14 I3 14 8 11 14 17 15 16 17 0.62 0.93 - 11 12 13 13 14 12 15 14 17 17 18 19 0.50 0.80 0.83 - 9 11 14 15 I 1 14 15 18 18 19 20 0.58 0.87 0.91 0.63 - 6 15 16 12 15 16 19 19 20 21 0.73 1.02 0.98 0.78 0.39 - 14 15 14 17 15 18 18 19 20 0.77 0.93 0.94 0.98 1.05 1.01 - 9 7 8 7 10 10 11 12 0.89 1.08 1.07 1.11 1.18 1.11 0.65 - 8 11 8 11 11 12 13 0.57 0.59 0.91 0.78 0.86 1.01 0.50 0.62 - 5 8 I1 9 10 11 0.82 0.84 1.16 1.03 1.11 1.26 0.58 0.88 0.39 - 7 10 8 9 10

0.89 1.07 1.06 1.10 1.18 1.10 0.49 0.64 0.62 0.54 - 3 3 4 5 1.13 1.31 1.31 1.34 1.42 1.35 0.72 0.88 0.86 0.78 0.23 - 5 6 7 1.13 1.15 1.32 1.35 1.43 1.36 0.73 0.89 0.70 0.62 0.23 0.39 - 1 2 1.21 1.23 1.40 1.43 1.51 1.44 0.81 0.97 0.78 0.70 0.31 0.47 0.08 - 3 1.25 1.26 1.43 1.47 1.55 1.47 0.84 0.99 0.81 0.73 0.34 0.50 0.11 0.19 -

regard to the phylogenetic clustering in Figure 2. M. muscuZus with domesticus types of mtDNA are indicated by mu in parentheses.

“Samples 1-34, 38-40, 87, 88. ’Samples 45-48. ‘Wild mice from California (sample 52) and Indiana (samples 55-57).

the two trees. The 13 lineages form subsidiary branches or twigs attached to the major branches that were evident in Figure 2. No new major lineages were uncovered by this second survey, i.e., none fall outside the cluster of lineages established by the first survey.

~ h y ~ o g e n e ~ ~ c a ~ l ~ inferred ~ u t a t ~ ~ n s : These trees (Figures 2 and 3) permit esti- mation of the number of mutations required to account for the variation observed at each site. These numbers for Figure 2 are summarized in Table 5. Most of the sites require only one substitution to account for the variation observed, but a few require more events. Examples of these hypervariable sites occur in the D-loop and in the unidentified reading frames, enhancing estimates of the variability of these regions.

Geographic structuring: The trees in Figures 2 and 3 are also notable from a

694 FERRIS ET AL.

dumesticus branch

1

Laboratory (1-34, Strains 38.39)d Ohio (40 )d California (49) d Jopan (87,88) mu AKR/FuRdA (35) d NMRVNovy (36) d SL/NiA ( 3 7 ) d

I Pennsylvonio (41)d California (53,54) d Maryland (58) d California (50,51) d Yugoslavia (70,71) d

lrroel (42)d Switzerland (73) d Switzerland (77) d Egypt (66)d NZB etc. (45-47,48) d Colifornia (52) d Indiana (55-57) d Egypt (67) d Morocco (68) d

rg 1

Peru (59-64) d Yugoslovia (72) d Italy (76) d Denmark (78) mu Oenmork (79,801 mu Israel (65)d Morocco (69) d

Wales (44)d I taly (74,751 d

4 5

2 Colifornio (431 d

costuneus branch Y I Thailand (89) c

Jopan (85,861 mo 5

musculus 6 Yugoslovio (83,841 mu 10 Czechoslovokio (81) mu

branch Czechoslovokio (82)mu

30 25" 10 5 0

Average Number of Substitutions per Lineoge

FIGURE 2.-Evolutionary tree of mitochondrial DNAs from commensal mice. The country or state of origin of the mice is given, followed by the sample number (Tables 1 and 2) and letters denoting species identity according to protein electrophoresis and morphology. These are d , mu, ino and c, respectively, for M. domesticus, musculus, nzolossiizus and custaneus. The appearance of m u in the domesticus branch of the tree is due to introgression (FERRIS et al. 1983b). Samples indicated in boldface lettering are inbred strains. In four instances the individual strains themselves are given, because the place of origin is not known (4 Table 1 and DISCUSSION, also regarding the other inbred strains). Those strains labeled Ohio, Japan and Wales have a mtDNA type attributed to contamination-cJ: FERRIS, SAGE and WILSON (1982) and DISCUSSION. The tree was constructed by parsimony methods (FERRIS, WILSON and BROWN 1981; FERRIS et al. 1983a,b) from the results in Table 3 and APPENDIX, Tables 1A-12A. Relationships within the domesticus branch of the tree were derived from the 60 variable sites among the 25 domesticus mtDNAs in Table 3; 24 sites were phylogenetically informative. The most parsimonious tree, which is shown, required a total of 73 point mutations at the 60 sites. Relationships within the musculus branch were determined similarly, considering 25 variable sites (of which seven were phylogenetically informative) and using the donwsticus mtDNA of known sequence as an outgroup. The number of point mutations inferred to have occurred along each lineage is indicated. A fragment method was used to determine the


geographic standpoint. Apart from the tendency for mouse mtDNAs from Egypt to cluster in the tree (see Figure 2 and especially Figure 3), macrogeographic structuring is not strongly evident in the Old World, where M. domesticus is a native species. For example, mice from opposite ends of the Mediter- ranean (Morocco and Israel) have nearly identical mtDNAs whose genealogical relationship to the intervening Egyptian mtDNAs is fairly remote. In the New World, to which M. domesticus was introduced within the past few hundred years, there is also no apparent macrogeographic structuring. The mtDNA types found in North and South America are fairly representative of the types found in Europe and the Mediterranean region.

Microgeographic structuring is strongly evident, however, in both the New and Old Worlds. Of the 25 localities at which more than one mouse was sampled (Tables 2 and 4), 20 were apparently homogeneous. If domesticus mtDNAs were distributed randomly, at most two of the 25 population samples would have been expected to be homogeneous. Probably, therefore, some of these homogeneous populations were founded by a single female.

In the three heterogeneous localities in California, namely, Buellton, Napa and Antioch, none of the multiple types is unique to a locality, and the types there do not form a monophyletic group with respect to other mtDNA types. This means that the multiplicity at Californian localities almost certainly did not arise in situ; rather, it is attributable to multiple founders. By contrast, in the two heterogeneous Egyptian localities, some types, such as DE, may be unique to a locality and could have arisen in situ (see Figure 3), but too few mice were examined to prove this.

Our survey also demonstrated the temporal persistence of particular mtDNA clones at four localities in California: AA in Buellton in 1980 and 1981, AB in San Pablo from 1979 to 1980, AC in Napa in 1979 and 1981 and CH in Petaluma in 1980 and 1981 (see Tables 2-4). In addition, we note the occurrence of the JN type in Antioch, 60 km from Corte Madera, where the foun- dress of the SF/Cam strain was trapped in 1951 (SAGE 1981b). This mtDNA type has probably persisted in the San Francisco Bay Area for 30 years.

Other MtDNAs Many sites were mapped by the sequence comparison method in mtDNAs

from additional mice belonging to species other than Mus domesticus; the frag-

relationships of the three main branches to one another and the location of the roots within the doinesticus and musculus branches; we considered the 199 phylogenetically informative and 249 uniquely variable fragments among 31 types of mtDNA--i.e., all those in Table 3 except M. ceruicolor; M. spretus, thus, served as an outgroup. The distances among the three types of commensal mice were based also on the percent sequence differences calculated in Table 7 (CJ: also Figure 5) . We have found by parsimony analysis that the restriction maps reported by YONEKAWA et al. (1981, 1982) are consistent with this branching order. In view of the great distance of the M. spretus outgroup, simultaneous loss of the same fragment in different taxa due to the occurrence of different point mutational events, the presence of only one mouse in the castaneus branch and the difficulty of determining fragment identities between distance taxa, w e regard the branching order among the three commensal lineages (namely, domesticus, castaneus and musculus-molossinus) as tentative.

696 FERRIS ET AL.

A A California, England 0 A 0 Denmark - : A'} California sz 0 XZ England

AY Scotland AB California AT SL/NiA BA Maryland

California, Scotland, Yugoslavia Scotland

I EF FM F I VB' uc' CE DE cc CD TW CH

Morocco

Israel Switzerland Egypt Switzerland, Italy

Egypt, us.

RC California, Peru H K Yugoslavia I L Italy, Scandinavia I - i : KL}Germany I A'

GJ Israel, Morocco JN California YV Italy i

I I I I 3 2 1 0

Average Number of Substitutions per Lineage

FIGURE 3.-Evolutionary tree for domesticus mtDNAs based on cleavage maps for two restriction enzymes. T h e tree was constructed by a parsimony analysis of the 38 variable sites recognized by MboI and Hinfr (6 Tables 3 and 4 and APPENDIX, Tables 11A and 12A), of which 19 were phylogenetically informative. A total of 52 point mutations was required to derive these mtDNAs from a common ancestral mtDNA, including an unidentified one along the VB' lineage (6 footnote ' in Table 1 IA). T h e letters at each lineage refer to the MboI and Hinfr patterns, respectively. Those lineages terminated by solid circles represent lineages not included in Figure 2. There is congruence with the domesticus section of the tree in Figure 2 except for a shift in the EF lineage (mouse 68 in Figure 2) and a minor change in the relationship of the J N and YV lineages (mice 43, 44 and 74, 75 in Figure 2).


ment patterns for these mtDNAs appear in Table 3. In addition, the mtDNA of M. spretus was mapped by the double-digestion method (Figure 4). In all eight species the length of mtDNA appeared to be 16.3 & 0.1 kb, no significant interspecific differences in length being detected. Moreover, the distribution of variable and constant sites was fairly similar to that observed for domesticus mtDNAs, as was the percentage of transitions and silent substitutions.

In the case of mice whose mtDNA is highly divergent from the mtDNA of known sequence, some of the restriction fragments could not be mapped with much certainty by the sequence comparison method. For this reason, our estimates of degrees of sequence divergence among these mtDNAs are based also on the fraction of fragments in common (Table 7). For the spretus-domesticus comparison it was possible to make divergence estimates based on map comparisons as well. In this case, the map method gives a higher estimate (16.7%) than the fragment method does (8.4% based on the 1 1 enzymes listed in Table 3 and 13.0% based on the 12 enzymes used to prepare the maps in Figure 4). Such a result would be expected because the fragment method does not correct for back mutation or heterogeneity among sites as regards proba- bility of cleavage. This inadequacy of the fragment method is serious only

Mus domesticus I

e e a c ffaaf c h h cgeejb yhebfmb y m d y e I1111 I 'I1 Y II \\If 19 I I

Mus spretus

0 4 8 12 16 Kilobases

FIGURE 4.-Cleavage maps for mitochondrial DNA of a house mouse, M. domesticus, and an aboriginal mouse, M. spretus. Both mtDNAs were subjected to single and double digestion with 12 enzymes recognizing six-base sequences and designated on the maps with a single-letter code (see MATERIALS AND METHODS). Three additional enzymes tried (SmaI, SalI, K ~ J z I ) cut neither mtDNA. The precise locations of the domesticus sites are plotted in accordance with the known sequence of this mtDNA (BIBB et al. 1981). The sites in spretus are based on use of the sequence comparison method in conjunction with the double-digest results. These linear maps of the circular mtDNA start at the same point as does the 16295-bp domesticus sequence of BIBB et al. (1981), with the first base of the tRNAPhe gene corresponding to 0 kb on the maps (see Figure 1). Each map has 31 cleavage sites, the site at 13551 bp being recognized by both XhoI and Ana1 in domesticus.

698 FERRIS E T AL.

TABLE 7

Quantitative comparison of fragineut patternsfor mouse mtDNAs

Mice compared 1 73 76 81 82 83 85 89 90 91

1 M. domesticus 73 M. domesticus 76 M. domesticus 8 1 M. musculus 82 M. inusrulus 83 M. musculus 85 M. inolossinus 89 M. castnneus 90 M. spretus 91 M. ceruicolor

33 0.81 - 1.20 1.61 3.35 3.89 3.74 4.03 3.36 3.91 3.46 3.96 3.38 3.72 8.41 8.46

-

11.51 11.31 I

47 60

3.55 4.02 3.58 3.72 3.54 8.84

10.07

-

113 126 114

0.87 0.65 0.62 4.06 9.05

11.62

-

126 133 127

37

1.01 I .08 4.52 9.2 1

1 1.90

-

112 125 115 27 42

0.76 4.30 8.70

11.64

-

116 128 120 26 45 29

3.92 9.06

1 I .25

-

115 211 124 213 116 213 135 226 147 231 139 218 132 226 - 235

9.89 13.77 10.03 -

234 239 22 1 239 244 236 235 223 260 -

~ ~~ ~~ ~~ ~ ~~ ~ ~ ~ ~ ~

The upper right half of the matrix gives the number of fragment differences among ten different mtDNAs of six species which were digested with 1 1 enzymes (Table 3). Estimates of the percent divergence in nucleotide sequence calculated with equation 20 of NEI and LI (1979) appear in the lower left portion. For comparisons vs. M. ceruicolor, the percentages are based on the six- and four- base enzymes only, as no other taxon shared any fragment produced by the five-base enzyme AvaII. The fragment method of comparison gives lower estimates of degree of sequence difference than the site method used in Table 6 except in the case of small differences [cf. also NEI and LI (1979) and GEORGE ( 1 982)]. Within the domesticus and musculus-molossinus groups, the two methods give comparable results (see Table 6). When domessticus, musculus-molossinus and castaneus are compared with one another with the site method, the values are on the average 0.6% higher than tabulated here. When the site method could be applied to comparisons involving M. spretus and M. ceruicolor, the values were about 2 and 4% higher, respectively. The fragment results are shown here, as only this method could be applied to all taxa, due to the inability to map completely the non-domesticus types of mtDNA (4 APPENDIX).

when the sequences compared have diverged by more than 5% (FERRIS et al. 1983b).

The average divergence between mtDNAs from the musculus mice (0.92%) is comparable with that within the domesticus mice. In contrast, there may be little variation in M. molossinus: mtDNAs from two mice (no. 85, 86) were identical with 11 enzymes even though they come from islands 700 km apart. YONEKAWA et al. (1981, 1982) also detected no differences at 22 sites among at least 46 molossinus mice from 15 localities in Japan.

It is notable also that molossinus mtDNA is more similar to those of musculus than to those of other commensal species. This result, based on about 167 restriction sites per genome, is in agreement with that of YONEKAWA et al. (1981, 1982).

Figure 2 shows genealogical relationships among the mtDNAs of the commensal mice examined in this report. The domesticus cluster of mtDNA lineages stands out clearly as a genealogical unit that has no overlap with the two other major lineages of commensal mouse mtDNAs. The tree analysis also suggests that castaneus mtDNA is more related to domesticus mtDNA than are the mtDNAs of musculus and molossinus, but an alternative tree, in which there is a three-way split between domesticus, castaneus and musculus-molossinus lineages (Figure 5), fits nearly as well with the data. Figure 5 also depicts the probable relationships of the M. spretus and M. cemicolor mtDNAs to those of the com-


Millions of Years 4-8 3-6 2-4 1-2 0

I I I 1 I I I I 1

699

Origin of commensal mice domesticus

custuneus

musculus, molossinus

: cervicolor

I I 1 I 1 I I I I 16 12 8 4 0

Percent Sequence Divergence

FIGURE 5.-Evolutionary tree for the mitochondrial DNAs of eight species in the subgenus Mus. The thick lines lead to the four commensal species. The three-way split shown among the domesticus, castaneus and musculus-molossinus lineages fits with the mtDNA data almost as well as does the branching order shown in Figure 2. The extents of sequence divergence are based on those shown in Table 7 as well as on values obtained by comparing restriction maps (partial and complete, see legend to Table 7) and should be considered as approximations. The time scale comes from assuming a rate of mtDNA evolution of 2-4% per million years (BROWN et al. 1982; FERRIS et al. 1983b). M. abbotti and M. hortulanus are connected to this diagram with dashed lines on the basis of preliminary studies with two to seven restriction endonucleases. From the magnitude of the differences, these two taxa are about as different from the commensals as is M. spretus. Further- more, M. abbotti and M. hortulanus appear almost as different from each other and from M. spetus as all three are from the commensals. The clustering of the aboriginal species shown is based on a parsimony analysis of 14 phylogenetically informative sites in the XbaI and AvaII maps, of which seven sites bear on this question.

mensa1 mice. Finally, we show with dashed lines the possible phylogenetic relationships of the mtDNAs from M. abbotti and M. hortulanus, which have been subjected to preliminary restriction analysis only.

DISCUSSION

Genetic classajication of wild mice The mitochondrial tree (Figures 2, 3 and 5) generally reinforces the genetic

classification of mice introduced by SAGE and MARSHALL (SAGE 1981a; MAR- SHALL and SAGE 1981; MARSHALL 1981). It emphasizes not only the distinctiveness of M. domesticus from the other commensal species of mice but also the inadequacy of the old classification which postulates the existence of three

700 FERRIS ET AL.

subspecies in Western Europe and the Mediterranean area. Neither the protein nor the mtDNA data lend any credence to the genetic reality of these subspecies-domesticus in the north, brevirostris in the central and praetextus in the south part of the geographic region. Consider the mice from Italy, Yugoslavia and Azrou in Morocco, previously called brevirostris, and the so-called praetextus mice from Israel, Egypt and Erfoud in Morocco. In Figures 2 and 3 these former subspecies are interspersed in various parts of the M. domesticus tree and do not cluster separately from the mice of Switzerland, the British Isles and Germany, which were referred to as M. m. domesticus under the old system (SCHWARZ and SCHWARZ 1943). Thus, the mtDNA results, like the protein electrophoretic data (SAGE 1978, 1981a; BRITTON and THALER 1978; MAR- SHALL and SAGE 1981; BONHOMME et al. 1983), provide no genetic evidence for taxonomic subdivisions within the species now designated M. domesticus. We, therefore, recommend that the practice of distinguishing between M. d. domesticus and M. d. brevirostris (e.g., GROPP et al. 1982; YONEKAWA et al. 1982) be discontinued.

The mtDNA tree is also in agreement with the SAGE and MARSHALL view that the three European types of aboriginal mice-spretus, hortulanus and abbotti-lie well outside the commensal group of mice and that these three species differ substantially from one another genetically.

Further work is needed to clarify relationships among the species of commensal mice. According to our mtDNA results and those of YONEKAWA et al. (1 98 1, 1982), molossinus and musculus are remarkably similar. Indeed, it would appear from our results that molossinus mtDNA is derived from one of the musculus lineages (Figure 2). Yet the protein electrophoretic results suggest that molossinus is about as similar genetically to castaneus as to musculus (SAGE 1981a; R. D. SAGE, E. M. PRAGER and A. C. WILSON, unpublished results and calculations). A more comprehensive nuclear and mitochondrial study of relationships between molossinus, musculus and castaneus mice from numerous parts of their geographic ranges is thus in order.

Time scale fo r mouse evolution The time scale in Figure 5 is based on the assumption that rate of point

mutational divergence in mtDNA is between 2 and 4% per million years. This is the rate inferred from mtDNA comparisons made with hominoids (BROWN, GEORGE and WILSON 1979; FERRIS et al. 1981; BROWN et al. 1982), goats and sheep (UPHOLT and DAWID 1977; BROWN, GEORGE and WILSON 1979), Old World monkeys (GEORGE 1982) and species within the horse genus (M. GEORGE, JR., personal communication), whose divergence times are known approximately from the fossil record. What evidence is there for a similar rate of divergence in mice? Consider, first, the rates of mtDNA divergence relative to that of nuclear DNA. As pointed out by SARICH (1972), WILSON, CARLSON and WHITE (1977) and SARICH and CRONIN (1980), the absolute rates of nuclear divergence are similar for primates and rodents. We also know that the rate of mtDNA divergence is five to ten times higher for mtDNA than for nuclear DNA in both mice and primates (FERRIS et al. 1983b). Hence, the


absolute rate of mtDNA divergence in rodents is probably similar to that for primates, i.e., 2-4% per million years. This view receives support from the fossil evidence suggesting that spretus-like mice lived in North Africa about 4 million years ago (MARSHALL 198 1).

The possibility that mtDNA divergence could be faster in mice than 2-4% per million years will occur to readers familiar with the evidence that Northern Europe was glaciated and uninhabitable by house mice until about 8000 years ago (KERR 1983).3 Yet, in Scandinavia, northern Germany and Scotland, we find apparently unique, derived types of mtDNA. If all these variant types originated in situ within the past 8000 years, it would be necessary to invoke a higher rate of mtDNA divergence than was assumed before.

Our reason for tentatively rejecting this possibility can best be understood by considering the Scandinavian and northern German cluster of lineages in Figures 2 and 3. Comparatively late in this survey, we discovered a very closely related type of mtDNA in the mountains of central Italy, at Cittaducale (mice 76 and 147-149). What we thought of as a northern clade had suddenly grown southern roots (see Figures 2 and 3 and FERRIS et al. 1983b). Clearly, the possibility exists that the northern clade, including the derived types, arose in southern Europe in preglacial times and moved north as the ice retreated. Hence, we regard the attempt to infer rates of mtDNA evolution from histor- ical biogeography as premature, pending a more thorough geographic survey of mtDNA variability. For this reason, we prefer the estimate of 2-4% divergence per million years for mtDNA, which is based ultimately on fossil evidence.

Such an estimate implies that the first commensal mice may have begun their association with the hominid lineage at the time indicated by the arrow in Figure 5, i.e., 1-2 million years ago. This implication fits with the data inferrable from SAGE’S (1 98 la) protein comparisons and contrasts with the old view that the commensal association of mice with people began when agricul- ture started (SCHWARZ and SCHWARZ 1943). Fossil remnants of mice from early archaeological sites approximately a million years old also support the hypoth- esis of a preagricultural origin for commensal mice (BROTHWELL 1981; TCHER-

Further geographic consideration5 The low tendency for macrogeographic structuring of mtDNA variability in

house mice is consistent with their commensal ties to the highly mobile human species. MtDNA variability among humans also exhibits little geographic structuring according to CANN, BROWN and WILSON (1982), CANN (1982) and CANN and WILSON (1983), who made a worldwide survey of 163 variable restriction

NOV 1983).

’ HAUSWIRTH and LAIPIS (1982) have observed an anomalously high rate of mtDNA “divergence” within a herd of domestic cows, apparently due to segregation of a mixture of mtDNAs in the germ line-rather than to the occurrence of new point mutations. The observed stability of the old inbred type of mtDNA among the sublines of major strains of laboratory mice for about 50 years (i.e., more than 200 generations) implies that this phenomenon may not occur in mice. Examples include DBA/PN us. DBA/LiA, BALB/cJ us. BALB/cByA and BALB/cCdA and C57BL/6J us. C57BL/LiA us. C57BL/ImrHeA. Additional examples of mtDNA stability over hundreds of generations in the mice could also be cited from our comparative studies of both laboratory and wild mice.

702 FERRIS ET AL.

sites and 14 kinds of length mutations in human mtDNA. More geographic structuring is evident in those rodent species whose distribution has not been homogenized by people. Thus, AVISE, LANSMAN and SHADE (1979), AVISE et al. (1979) and LANSMAN et al. (1983) concluded that divergence in mtDNA sequence was proportional to geographic distance within species of deer mice and gophers. A strong geographic component was also found in a survey of mtDNA variability within ape species (FERRIS et al. 1981).

Model for Lhromosomal evolution The mtDNA results favor one model for explaining the remarkable tendency

for isolated populations of M. domesticus in mountainous regions and archipe- lagos to undergo chromosomal fusions (SAGE 1981a; ADOLPH and KLEIN 1981; GROPP et ~1.1982; NASH, BROOKER and DAVIS 1983), reducing the haploid number of chromosomes to less than the normal value of 20. According to this model, the fixation of a karyotypic mutation that causes meiotic problems in the heterozygous state is most likely to take place when a population is small. This is true even when the heterozygote disadvantage is minor (WILSON et al. 1977; LANDE 1979), as it is for mouse chromosomal fusions (GROPP et al. 1982). Mouse populations are likely to have had few members not only when small islands or mountain valleys were being colonized after the Ice Age but also during later bad times. The effective number of maternal lineages in populations with such a history of repeated bottle-necking would be very low. Furthermore, one might expect any population that had fixed several fusions to be so isolated meiotically from subsequent, karyotypically normal, immi- grants as to reduce the inflow of additional mtDNA types markedly.

The mtDNA results are consistent with this expectation. Our survey included the following six populations that have become fixed for fusions, the number of fused chromosomes per haploid genome being given in parentheses: Westray (Z), Zadar (6), Poschiavo (7), Tirano (8-R. D. SAGE, unpublished ob- servation), Milano I1 (8) and Cittaducale (9). Westray, with only two fusions, is the only case in which more than one mtDNA type has been found (Tables 3 and 4). If a more comprehensive survey were to confirm the apparent mtDNA homogeneity of the populations that have fixed at least six fusions, it would be consistent with a recent origin for these populations from a few founders.

In support of the view that such populations may be young, we note the low degree of mtDNA divergence between some of these populations and karyotypically normal mice. The Cittaducale population, for instance, differs in mtDNA by only 0.08% from the Hov mice (Table 6); this corresponds to a divergence time of 20,000 to 40,000 years. Hence, the average rate of fixation of fused chromosomes may have been approximately one per thousand years in the Cittaducale population-an estimate that is similar to others not based on mtDNA comparisons (SAGE 1981a; NASH, BROOKER and DAVIS 1983). The fact that this rate exceeds those estimated for other mammalian species by a factor of at least 10 (BUSH et al. 1977; WILSON et al. 1977) intensifies interest in understanding this phenomenon.


Origtns of laboratory mice High frequency of one type of mtDNA: Our study confirms that M . domesticus

females gave rise to virtually all of the common laboratory strains (YONEKAWA et al. 1982) and that most of these strains (39 of 50) have the “old inbred” type of mtDNA (FERRIS, SAGE and WILSON 1982), whose frequency in the wild domesticus mice surveyed is only about 0.04 (see Table 4). Seven other mtDNA types are found in laboratory strains, four closely related to the old inbred type and three being at remote points in the genealogical tree for domesticus mtDNAs (Figure 2).

To explain the predominance of the old inbred mtDNA in the first 14 laboratory strains tested, FERRIS, SAGE and WILSON (1 982) divided the strains into two groups: (1) nine old inbred strains, established before 1922, apparently from five or more females, most or all of which came from the pet mouse trade (see Table 1) and (2) five “new inbred” strains, established from five females caught since 1950 at known (and diverse) places in the territory occu- pied by wild domesticus mice. All strains in group 1 had the same mtDNA type, despite exhibiting large interstrain differences at the nuclear gene level, but only one strain in group 2 had this type.

The result for group 2 was consistent with geographically random sampling, whereas the result for group 1 was not, since the chances of picking five mice with the old inbred type from a species in which this type has a frequency of 0.04 is (0.04)5 or lo-’ (FERRIS, SAGE and WILSON 1982). Three possible ways of explaining this result were considered: (1) nonrandom sampling, i.e., sampling from a population in which the old inbred type was already at high frequency, (2) a selective advantage in the laboratory for mice with the old inbred type and (3) interstrain contamination.

The 50 laboratory strains now surveyed confirm and extend the picture presented by FERRIS, SAGE and WILSON (1982). This can be seen by dividing these strains into categories A, B and C (as in Table 1). The A category contains 21 strains (nos. 1-20 and 35, Table 1) belonging to the old inbred group according to genealogical records (POTTER and KLEIN 1979; FERRIS, SAGE and WILSON 1982). Only one (no. 35) of these 21 strains lacks the old inbred type; instead, it possesses a very closely related type of mtDNA. In the B category there are 22 strains whose ancestors have been in the laboratory for at least 50 years. Most of these B mice probably have a connection with mice in category A, because they were derived from the pet mouse trade in northern Europe or, more immediately, from the outbred “Swiss” stock, which gave rise to one of the old inbred strains, SWR/J (LYNCH 1969; RICE and O’BRIEN 1980; HILKENS et al. 198 1). Only CE/JA seems to have been derived from a wild mouse at a known locality (in Illinois, in 1920). Sixteen of the B strains have the old inbred mtDNA, two have types closely related to it and four have the distantly related type characteristic of NZB mice. The third category, C, is for the seven strains established in the 1950s and 1960s from known localities in the wild. Three have the old inbred type, one has a closely related variant and the other two types of mtDNA are distantly related.

Contamination undoubtedly accounts for the presence of old inbred mtDNA

704 FERRIS ET AL.

in the two strains of molossinus (KL/oci and MOL3/JA) because the parental strain (mouse 86) retains the authentic molossinus type of mtDNA (Table 3); moreover, the old inbred mtDNA does not occur in the more than 40 wild molossinus screened from numerous parts of Japan (YONEKAWA et al. 1981, 1982). Contamination probably also accounts for the presence of “old inbred” mtDNA in MOR/Cv (FERRIS, SAGE and WILSON 1982), NZB/Ibm (FERRIS et al. 1983a) and NZB/BlPt (HUSTON et al. 1983). This phenomenon has also been invoked to explain the presence of the NZB type of mtDNA in NMRI/ Bom (FERRIS et al. 1983a) and of the SF/Cam/J type in SK/Cam/JEi (FERRIS, SAGE and WILSON 1982). Given the known and suspected high incidence of interstrain contamination by mtDNA, it is important to ask whether the presence of the old inbred type in CE/JA is due to contamination by the generally older, more abundant, and more successful (?) lines in categories A and B.

We are left with the problem of explaining the extremely high incidence of old inbred mtDNA in mice from the pet mouse trade. The pet population could have had the old inbred type at a high frequency because of drift (or selection) in this population. Alternatively, the high frequency could be the result of the pet mouse population being drawn nonrandomly from the wild, i.e., to a large extent from a particular locality where the old inbred mtDNA was at high frequency. The only locality in the native range of M. domesticus where indications exist that this type of mtDNA occurs is at Bunwell in Eng- land.4 Since northern Europe was the world center for pet mice in the last century, wild female mice from there could be responsible for the preponder- ance of the old inbred type of mtDNA in laboratory mice. This possibility can be investigated by further mtDNA surveys in Europe.

New mutations in the laboratory? What is the chance that mutant types of mtDNAs have arisen in the laboratory? The best opportunity to answer this question comes from considering the three strains (nos. 35-37) having mt- DNAs that are derivable from the old inbred type by a single detectable base pair change (see Table 3 and APPENDIX). None of these strains has a documented wild origin within the past 60 years (Table l) , and their types of mtDNA have not been found outside the laboratory. We approach this problem by taking account of the 36 strains in categories A and B that contain the old inbred type of mtDNA. Suppose that these 36 lineages and the three divergent types stem from a single female ancestor with the old inbred type of mtDNA and that the average divergence time between all possible pairs of the 39 lineages is 60 years. We are, thus, looking at 2340 years available for mtDNA divergence, during which time the expected number of base substitutions is 0.4 to 0.8 among all 39 lineages, assuming the standard divergence rate of 1-2% per million years per lineage (see DISCUSSION, Time scalefor mouse

Let us assume, further, that the restriction method detects 10% of the changes occurring in mtDNA, rather than just the 4.5% represented by the restriction sites examined. From the Poisson distribution we calculate a 3.8-7.4% chance of finding one mutant lineage among the 39 tested with the

‘An earlier publication (FERRIS, SAGE and WILSON 1982) reported old inbred mtDNA, via introgression, also in a mouse from Vejrumbo in Denmark. Subsequent investigation disclosed that the original wild M. musrulu5 stock was contaminated by breeding with inbred mice in the laboratory from which we received that sample.


restriction method. The chances of detecting two or three such mutant lineages, however, are 0.001-0.3%. It seems quite unlikely, therefore, that all three divergent mtDNAs arose in the laboratory.

The possibility that one of these three mtDNAs is of laboratory origin, although low, can at least be entertained. The most likely candidate is the mtDNA of strain 36 (i.e., NMRI/Navy) because it differs from the old inbred type by a C + T transition5 at a hypervariable site (see FERRIS et aE. 1983a, Tables 3 and 5 and APPENDIX). The same change has occurred independently along four different lineages in the tree for domesticus mtDNA (Table 5 and Figure 2). By contrast, the changes in strains 35 and 37 have each occurred only once in this tree.

We, therefore, suggest the mutations responsible for the mtDNA differences among strains 35, 37 (and probably 36) and the old inbreds preexisted in the mouse population from which laboratory mice were established. The situation is probably analogous to that for strain 4 1, whose ancestress was captured in the wild and whose mtDNA differs from the old inbred type at one restriction site (Table 3 and APPENDIX, Table 12A).

This work was supported by the Miller Institute, University of California (S.D.F.), National Science Foundation grants DEB 78-02841 and DEB 81-12412 (to A.C.W.) and DEB 72-02545 (to the Museum of Vertebrate Zoology, R.D.S.) and National Institutes of Health grant GM-21509 (to A.C.W.). We thank all those listed in Tables 1 and 2 for providing mice and also P. ALBERCH, S. GABER, J. MEANS and I. SAVI? for help in collecting the wild mice. We are grateful to U. GYLLENSTEN for technical assistance, S. M. CARR and B. S. CHAPMAN for assistance with computer analyses and to B. E. L. TIMMONS for unpublished restriction maps. For helpful discussion we thank R. J. BERRY, R. L. CA”, S. M. CARR, P. CHALLINOR, D. A. CLAYTON, K. FISCHER LINDAHL, A. GROPP, U. GYLLENSTEN, M. HAMMER, J. HILGERS, V. M. SARICH and H. YONEKAWA.

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Corresponding editor: M. NEI

APPENDIX

Table 1A lists the sites for the 11 restriction endonucleases employed in this study in the mouse mtDNA of known base sequence (BIBB et al. 1981). All of our A fragment patterns were entirely in agreement with those predicted from the sites in Table 1A for this mtDNA.

Tables 2A-12A list the variable sites inferred from cleavage maps constructed by the sequence comparison method (MATERIALS AND METHODS) for each restriction enzyme. (Double digests of M. sprefus mtDNA-Figure 4-aided in location of the Hirid111 C and XbaI E sites.) T h e base changes listed in Tables 2A-12A indicate the change on the light strand. When it is possible to determine for protein-coding genes the nature of the change, we indicate, after the listing of the location and base interconversion involved, silent and replacement substitutions by S1 and S3 and by R1, R2 and R3, where the numbers refer to codon positions. In each table the fragment patterns and their associated maps are referred to by capital letters; those designated ‘dornesficus” are derived from among samples 1-80, 87, 88 and 92-204 in Tables 1 and 2 of the main text; those designated “others” are derived from samples 81-86, 89-91 and 205 (4 Tables 3 and 4 of the main text). In some cases of the enzymes HindIII, HiucII, Arc1 and AvaII, patterns of the dornesticus type are seen also in M. rnusculus, molossirius and castaneus mtDNA. As it was not always possible to infer complete cleavage maps for the non-domesticus mtDNAs, in those instances we list in the lower portions of the tables the sizes in base pairs of the fragments produced and the locations of sites inferred in partial maps (Tables 2A and SA-12A). Constant as well as variable sites are included for the partial maps. From Tables 2A-12A used in conjunction with Table IA, it is possible to reconstruct readily the agarose and polyacrylamide gel patterns obtained for each pattern with each enzyme.


I t should be remembered that the methodology employed, the close locations of two or more semisites and, in the mtDNAs most distantly related to the known sequence, the possibility of more than a single base change leading to generation of a restriction site make it difficult to infer with certainty the locations of all sites in the non-domesticus types of mtDNAs.

TABLE 1A

Locations of restriction sites for 11 endonucleases in the mouse mtDNA of known base sequence

Hind1119136 11081 11969 XbaI612 953 8529 8984 10907 15973 HincIl364 1858 5123 5452 7718 AccI5709 5754 6222 7035 9339 9598 14646 Anal178 424 2083 2357 2871 3480 3959 6005 12515 14239 FnuDIl317 1772 2007 3410 4972 11406 13039 14705 HpaII 133 1870 2519 3332 3400 5725 6112 6274 6388 8400 15736 Hue11184 1053 1606 2004 2215 2856 3335

3402 3871 4007 4129 4232 4670 6621 6749 7015 7541 7592 8182 8742 8811 9756 11118 11192 11526 12378 12417 12450 12666 14708 15156 15189 15739 15801 15923

Tup1634 2407 2429 3343 5220 5233 5438 6841 6885 7668 9577 9835 12080 13552 13562 14348 14620 15253 15850

Mbol 1889 2350 2438 2505 3103 3223 3566 3597 4065 4276 5884 5988 6328 6697 6731 6990 7085 7614 7677 8165 8937 9238 9385 10729 11168 11323 12028 13400 13592 13814 13919 14452 14749 15330 16175

HinfI 232 729 1367 3360 4689 5635 6034 6514 6882 7367 7434 7973 8893 9211 9220 9432 9526 9574 9837 11571 11938 12082 12277 12520 13009 13225 14251 14668 15115 15994

TABLE 2A

Variable restriction sites and fragments of mouse mtDNA digested with Hindi1

Location of site Presence of site

domesticus Others

Site Start Base change Functional region A B C

+ + + +

- - 1 3623 3625, A-G, R2 URF 1 2 9136 9136-41 CO 111 + 3 11611 11614, A-C Ser tRNA 4 1 1969 1 1969-74 URF 5 + + 5 12863 12867, C-T, RI URF 5

- - -

- - -

Pattern Fragment sizes (bp) Sites in partial map

D 9600 6100 618 None

710 FERRIS ET AL.

TABLE 3A

Vuriable restriction sites nf " w e mtDNA digested with XbaI


doineslirus Others Functional

Site Start Base change region A B C D E F G

1 953 2 1322 3 4947 4 5288 5 8529 6 8984 7 9653 8 9921 9 15973

953-8 1322, G-T 4947, C-T, R2 5289, T-C 8529-34 8984-9 9653, C-T, S3 9926, G-A, R2

15973-8

12s rRNA 16s rRNA URF 2 Tyr tRNA ATP 6 CO I11 URF 3 URF 4L D-IOOP

+ + + + - + - + - + + - + -

+ - - + + + + + + - - - - -


TABLE 4A

Variable restriction sites of mouse intDNA digested with Hind1

Site

1 2 3 4 5 6 7 8 9

10 11


doiaestirus Others Functional

Start Base change region A B C G D E F

1858 1858-63 16SrRNA + + + + - + + 5123 5123-8 AsntRNA + + + + + + -

- + - 5754 5756, A-C", S3 CO I + -

+ - - - + - - -

+ - + -

+ -

- - - - - - - - - 6222 6224, A-C", S3 CO I

6507 6510, C-A, G, R1 CO I 7147 7147, A-G, S3 CO 11 8637 8642, T-C, S3 CO 111 - + - - + + +

- - - - - -

- - - - - - 12135 12136, C-T, R2 URF 5 12658 12659, C-T, S3 URF 5 15606 15610, T-A D-IOOP 16074 16074, C-G D-loop + - - - +

- - - - - - - - - - -

Mutation of A to either C or T results in generation of a Hind1 site. C was chosen because M. spretus (pattern E) was inferred to have an AccI site in the same location (pattern H in Table 5A).

71 2 FERRIS ET AL.


I I I I +

I I I I I

1 + 1 I I

I 1 + 1 I

I I 1 + 1

I I I I I

I I 1 + 1

+ + I + I

1 + 1 + 1

6

U

b h

4 0 2

714 FERRIS ET AL.

TABLE 7A

Variable restrictiotz sites of mouse inlDiVA digested with FnuDII


domPsticus Others Functional

Site Start Base change region A B H I D E F G

1 2 3 4 5 6 7 8 9

10

1772 3410 3583 4972 9505 9595

10592 1 1406 13039 14705

1772-5 3410-3 3584, A-G, S3 4972-5 9508, T-G, R2 9596, A-G, S3

10595, A-G, S3 1 1406-9

14705-8 13039-42

16s rRNA URF 1 URF 1 T r p tRNA URF 3 URF 3 URF 4 URF 4 URF 5 Cvto b

+ + + + - + - - + + + + + + - - - - - - - + - - + + + - + + - + - - + - - - - -

+ + + + + + - + + + + + - - - - + + + + + + - +


71 6 FERRIS ET AL.


I I 1 + 1 I

+ I I I + +

+ + I I + +

+ 1 + 1 + +

+ I I I + +

I I I + + +

I I I + + +

I I I + + +

I I I + + +

I I I + + +

I I I + + +

I I I + + +

718 FERRIS ET AL.

TABLE 11A

Variable restnctioiz sites mdfragmeiits of mouse mtDNA digested with MboI

Location of site

Functional

1 730 730,A-G 12SrRNA - - - - - - - 2 3566 3566-9 URF 1 + + + + + + + 3 3597 3597-600 URF 1 + + + + + + + 4 3922 3922,T-G,S3 URF 2 5 4033 4033,T-G,R3 URF 2 6 4187 4188,G-A,R2 URF 2 7' 4276 4276, G-A, T , C, S3 URF 2 + + - - + - + 8 5016 5019,A-C Ala tRNA - - + - - - - 9 5668 5668,C-G,R2 CO I

10 5988 5988-91 CO I + + + + + + - 11 6990 6990-3 AsptRNA + + + + + + + 12 7378 7378,A-G,S3 CO I1 13 7614 7614-7 CO I1 + + + + + + + 14 9187 9188,T-A,S3 CO I11 15 9793 9795,A-T,R1 URF 3 16 10840 10841, G-A, S3 URF 4 17 11001 11004, T-C, S1 URF 4 18' 11168 11168,G-A,T,C,S3 URF4 + + + + + + + 19 11323 11323-6 URF 4 + + + + + + + 20 12028 12028-31 URF 5 + + + + + + + 21' 12515 12516, G-A, RI URF 5 - + - + - - - 22 12955 12957, G-T, RI URF 5 23 13080 13082, C-T, S3 URF 5 24 13400 13400-3 URF 5 + + + + + + - 25 13814 13814-7 URF 6 + + + + - + + 26 13919 13919-22 URF 6 + + + + - + + 28 15218 15218, A-G, S3 Cyto b

Siteb Start Base change region A B C D E F G

- - - - - - - - - - - - - -

+ - - - - - -

- - - - - - -

- - - - - - -

- - - - - - - + - - - - - -

- - - - - - - - - - - - - -

- - - - - - - - - - - - - -

27h 14239 14240, G-A, T, C, S3 Cyto b - - - - - - - - - - - - - -

Pattern Fragment sizes (bp)"

P 1392 1280 1046 950 936 665 655 650 640 617 598 590 529 469 468 461 428 408 388 343 336 327 297 253 216 211 192 180 147 138 112 110 104 88 85 67 63 34 31

Q 1923 1720 1564 1491 845 842 772 705 680 655 618 598 488 469 442 439 354 301 297 242 222 123 120 112 88 67 63 44 34

Some revisions in the sites and fragments reported for patterns F, K, L, M, N and P in FERRIS ef a / . (1983b) are included.

'The variation in sites 4045 and 4065 reported in FERRIS et al. (1983a) was subsequently found to be in error. A site is invariably present at 4065. Therefore, the placement of the lineage corresponding to samples 45-48 in tree 2 but not tree 1 of FERRIS et al. (1983a) is consistent with the current report.

' Pattern V is identical with pattern U except for the presence of an additional, unexplained, 550- bp fragment.

In pattern M the fragment inferred to span the region from positions 9385 to 10729 (6 Table 1 A), a stretch of 1344 bp, has an apparent mobility in polyacrylamide gels equivalent to 1435 bp. ' Mutation of G to A at position 4276 results in the loss of the MboI site and the concomitant gain

of a Hili$ site at 4275 (site 8 in Table 12A). Mutation to either T or C at 4276 results in the absence of both sites, which is observed only in samples 42 and 135.


Presence of site" doarslirus Others

H I I K R S T U V X Y Z L Md N A' B'

Sites in partial maps

1889 2350 2438 2505 3103 3566 3597 4065 4276 5884 5988 6697 6731 7085 7614 7677 9238 9385 9793 10729 12028 13400 13592 13919 14452 14749 15218 15330 16175

2350 2438 2505 3103 3223 4065 5988 6697 6731 7085 7614 7677 8165 8937 9238 10729 11168 11323 12028 13592 13814 14452 14749 15218 15330 16175

'Mutation of G to A at position 1 1 168 results in the loss of the A4601 site and the concomitant gain of a Hili$ site at 11 167 (pattern K, site 18, in Table 12A), which among dontesticus-type mtDNAs is seen in sample 72. Mutation to either T or C at 11 168 causes the absence of both sites (6 pattern W in Table 12A); which among domesticus-type mtDNAs is seen in samples 129 and 137-139. The fragment patterns observed could also be explained by generation of a Hin$ site at 1 1168 by way of a silent change of C to T at position 1 1 17 1. However, a change to A or G at the same position to explain the absence of both sites would result in the substitution of Met for lie.

g See footnote to Table 6A. * See footnote to Table 6A.

720 FERRIS ET AL.

TABLE 12A

Vuriuble restrictioii sites uizdfrrrgvzents of mouse fntDNA digested with Hinfl

Location of site

Site Start Base change Functional

reeion A B C D E F

1 2 3 4 5 6 7b 8’ 9

10 11 12 13 14 15 1 6d 17 18‘ 19 20 21 22 23 24

232 1519 2999 3304 3360 3534 3784 4275 5435 5729 7973 8718 8965 921 1 9526 9574

10904 11 167 11255 12520 13225 14638 15115 16179

232-301-235-6 1523, A-C 2999, A-G, S3 3308, T-C, S3 3360-1 or 3363-4 3537, G-T, R1 3788, T-C

5435, A-G, S3

7973-401-7976-7 8718, A-G, RI

9211-2 or 9214-5 9526-7or9529-30 9577-8

4276, G-A, T , C, S3

5729, A-G, S3

8969, T-C, S3

10904, T-G, R3 11 168, G-A, T, C, S3 1 1255, T-G, S3 12520-1 or 12523-4 13225-6or 13228-9 14639, C-A, S3 15115-6or 15118-9 16 179, A-G

12s rRNA 16s rRNA URF 1 URF 1 URF 1 URF 1 Gln tRNA URF 2 CO I CO I ATP 6 CO 111 CO 111 CO 111 URF 3 URF 3 URF 4 URF 4 URF 4 URF 5 URF 5 Cyto b Cyto b D-IOOP

+ + - - - - + - _ - _ _

Pattern Fragment sizes (bp)”,’

P 2270 1993 1370 1026 880 855 638 533 505 489 485 480 447 420 417 367 362 345 339 318 243 216 212 202 188 100 90 88 77 74 67 66 52 24 9

Q

R

S 1993 1180 1026 980 920 910 770 736 638 570 497 489 485 480

As in P except delete 1993,855, 100 and 88, add 1655,657, 338, 198 and 183

As in P except add 946, delete 880 and 66

445 417 415 405 402 367 362 330 225 216 212 190 180 126 109 108 67 54 (9)

D’ 1993 1420 1370 1329 1026 879 750 565 533 505 485 482 480 447 417 405 367 339 330 318 295 276 243 216 212 202 195 143 77 67 9

E’ 1890 1610 1370 1230 1210 879 803 638 570 533 512 489 485 480 447 427 367 362 345 339 276 188 143 130 127 114 111 96 67 30 (9)

Some revisions in the sites and fragments reported for patterns E, 1, P, Q, R and S in FERRIS et ul. (198313) are included.

’See footnote to Table 10A. ‘See footnote to Table 1 1A.


Presence of site in domesticus mtDNA”

H I I K L M N O T U V W Y Z A ’ B ’ C ’

Sites in partial maps

232 729 1367 3360 4689 5569 5635 6034 6514 6882 7367 7434 8893 9211 9220 9432 11571 11938 12277 12520 13009 13225 14251 14668 15115 15970 15994

As in P plus 1705 and 15768

As in P except delete 5569

232 729 1367 3360 6034 6514 6882 7367 7434 7973 8893 9220 9432 9837 11571 11938 12520 13009 13225 14251 14668

232 1367 3360 4689 6034 6514 6882 7367 7434 8893 9211 9220 9432 9837 11571 11938 12082 12277 12520 13009 13225 14251 14668 15115 15994

232 729 1367 6034 6514 6882 7367 7434 11571 11938 12520 13009 14668 15115 15994

See footnote * to Table 10A. See footnote f to Table 1 1 A.

’See footnote to Table 10A.

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MITOCHONDRIAL DNA EVOLUTION IN