Copyright 0 1989 by the Genetics Society of America
Y Chromosome Evolution in the Subgenus Mus (Genus Mus)
Priscilla K. Tucker,' Barbara K. Lee and Eva M. Eicher The JacksonLaboratory, Bar Harbor, Maine04609 Manuscript received November 30, 1987 Accepted for publication January 20, 1989
ABSTRACT A 305 base pair DNA sequence isolated from the Y chromosome of the inbred mouse strain C57BL/10 was used to investigate the pattern and tempo of evolution of Y chromosome DNA sequences for five species in the subgenus Mus, including Mus spretus, Mus hortulanus, Mus abbotti, Mus musculus and Mus domesticus. Variation in hybridization patternsbetween species was character- ized by differences in fragment lengths of both intensely and faintly hybridizing fragments, whereas variation in hybridizationpatterns within species was characterizedprimarily by differences in fragment lengths of faintly hybridizing fragments. Phylogenetic analyses were conducted based on fragment size variation within and among species. Phylogenetic relationships inferred from these analyses partly agreewith the phylogenetic relationships obtained from biochemical and mitochondrial DNA data. We conclude that a set of DNA sequences common to the Y chromosomes of a closely related group of species in the subgenus Mus has evolved rapidly as reflected by sequence divergence and sequence amplification.
N the classical model of sex chromosome evolution, DNA sequences, AC11 (NISHIOKAand LAMOTHE I heteromorphic sex chromosomes are hypothesized 1986) and YBlO (EICHERet al. 1989), isolated from to have evolved from a pair of homologous chromo- the Y chromosomes from two inbred mouse strains somes by the suppression of crossing over between the hybridize to genomic DNA from males ofsome species sex chromosome precursors and the subsequent loss in the subgenus Mus and distinct hybridization pat- of gene function onthe sex chromosome that becomes terns characterized by differences in fragment length the Y chromosome (MULLER 1914). OHNO(1967) and hybridization intensity were observed for each recalled this hypothesis to explain vertebrate sex chro- species. These data suggest that Y chromosome-spe- mosome evolution including the evolution of the cific DNA sequences rapidly evolve in closely related mammalian X and Y chromosomes. The X and Y species. chromosomes of mammals exhibit extreme hetero- In this report we present additional evidence for morphism and, with rare exception,recombination the rapid evolution of DNA sequences found exclu- between them is limited to a small region (SOLARI sively on the mouse Y chromosome from an investi- 1974). Thus, the majority of the Y chromosome is gation of the tempo and mode of Y chromosomal monosomic and inherited from father to son. sequence change at both the species and population Recent molecular investigations of the human levels. We alsoinvestigated how a paternally inherited (GOODFELLOW,DARLING andWOLFE 1985) andmouse character contributes to an understandingof the phy- Y chromosomes (SINGH,PURWM and JONES 1981; logenetic relationships within the genus Mus. We used JONES and SINGH198 1;PHILLIPS et al. 1982; EICHER, pYB 10 (EICHERet al. 1989) as a probein combination PHILLIPSand WASHBURN 1983; LAMARand PALMER with eight restriction enzymes to identify species-spe- 1984; BISHOPet al. 1985; NISHIOKAand LAMOTHE cific patterns of hybridization from a large sample of 1986; NALLASETHand DEWEY 1986) identifiedDNA mice in the subgenus Mus, including Mus spretus, Mus sequences specific to or enriched on the Y chromo- hortulanus (= M. spicilegus, BONHOMME1986), Mus some. Additionally, using Y-specific or Y-enriched re- abbotti (= M. spretoides or M. macedonicus, BONHOMME peated DNA sequences as probes, restriction fragment 1986), Mus musculus and Mus domesticus. Throughout length polymorphisms have been identified among Y this study we recognize M. musculus and M. domesticus chromosomes of human populations (CASANOVAet al. as distinct species rather than subspecies or semispe- 1985) as well as among Y chromosomes of laboratory cies based on data from the hybrid zoneinvestigations inbred mouse strains and wild mice (LAMAR andPAL- of these two taxa (HUNT andSELANDER 1973; SAGE, MER 1984; BISHOPet d. 1985; NISHIOKAand LA- WHITNEYand WILSON1986; SAGEet al. 1986). MOTHE 1986; EICHER et al. 1989). For example, two MATERIALSAND METHODS ' Currentaddress: Museum of Zoology, University of Michigan, Ann Mice: Wild mice used in this study came from either Arbor, Michigan 48 109. established laboratory colonies or from field trapping. Spe-
Genetics 122: 169-179 (May, 1989) 170 P. K. Tucker, B. K. L.ee and E. M. Eicher cies, number of individuals sampled, original collecting lo- minimum of two laboratory raised mice from each taxa were calities and source of taxa used in our study are listed in analyzed. Table 1. DNA preparation: High molecularweight mouse ge- nomicDNA was prepared from either frozen tissue or RESULTS frozen nuclear pellets. DNA from liver, kidney, spleen, and testis was prepared following the method of JENKINS et al. Species-specific patterns of hybridization were ob- (1982). Frozen nuclear pellets were incubated at 65" over- served when Southern blots containing male genomic night in 10 ml of extraction buffer (50 mM Tris, pH 8.0, DNAs from M. abbotti, M. hortulanus, M. spretus, M. 100 mM EDTA, 100 mM NaCI, 1% SDS) and 0.5 mlof musculus, and M. domesticus (Table 1) were probed proteinase K (10 mg/ml) in 10 mM Tris, pH 7.5, prior to extraction with phenol following the method of JENKINSet with pYB10. A total of 180 different restriction frag- al. (1982). ments were identified by single digests of male ge- Restriction endonuclease digestions and Southern blot nomic DNA using eight different restriction enzymes, preparation: Restriction enzyme digests of genomic DNAs representing four (HaeIII, TaqI), five (Hinfl)and six were performed following the procedures of the supplier (BglII, EcoRI, HindIII, PvuII, PstI) base cutters. No BRL (Bethesda Research Laboratories, Inc.). Ten micro- grams of genomic DNA were digested for 4 hr and separated hybridization was observed when Southern blots con- by size in 1% agarose at 30 V for up to 20 hr. DNA was tainingfemale genomic DNAs from M. abbotti, M. transferred to Zeta-Probe nylon membranes (Bio-Rad) in hortulanus, M. spretus, M. musculus and M. domesticus 0.4 M NaOH overnight without pretreatment (REED and (Table 1) were probed with pYBlO (data not shown). MANN1985). This verifies the observation by EICHERet al. (1989) DNA labeling The probe pYB10, derived from the Y chromosome of C57BL10 (EICHERet al. 1989), was labeled that YBlO sequences are found exclusively on the Y with [a-"PI-dCTP (Amersham) following MANIATIS, chromosome in these five species. The hybridization FRITSCHand SAMBROOK(1982) using the T4polymerase kit patterns observed with PstI (Figure 1) typify all single supplied by BRL. The specific activity of labeled DNA for digestsusing theeight restriction enzymes listed all experiments was greater than 0.5 X lo9 cpm/pg. above.Three basic trendsemerged. First, there is Hybridization conditions and autoradiography: Nylon membranes were prewashed at 65" for 1 hr in 0.1 % SSC overall less hybridization toDNA from M. spretus and and 0.5% SDS and prehybridized at 65" for 4 hr in 100 ml M. hortulanus than toM. abbotti, M. domesticus and M. of 4 X SSCP [ 1 X SSCP = 121 mM NaCI, 15 mM Nan citrate, musculus. Second, forsix enzymes, HaeIII, TagI,BglII, 15 mM Na2HP04, 5 mM NaH2P04],10 X Denhardt's solu- HindIII, PvuII and PstI, at least one restriction frag- tion and 1% SDS. Membranes were hybridizedovernight at ment is shared amongall five species and these shared 65" in 20ml of 4 X SSCP, 2 X Denhardt's solution, 1% SDS, 0.2 pg/ml denatured sonicated salmon sperm DNA, fragments typically vary in intensity of hybridization and approximately 1.0 X lo6 cpm/ml denatured radioac- among species. Third,between speciesvariation is tively labeled probe. Following hybridization, the nylon characterized primarily by the presence and absence membranes were rinsed twicein 2 X SSC/O. 1% SDS at room of both intensely and faintly hybridizing restriction temperature for 15 min and three times in 0.1 X SSC/O. I % fragments, whereas within species variationis charac- SDS at 65" for 30 min. The stringency conditions at 65", terized primarily, but notexclusively, the presence 0.1 X SSC, and a 39% G-C content (determined from DNA by sequence analysis of YB10,EMBL accession number andabsence of faintlyhybridizing restriction frag- X 12900) were calculated to be approximately 98% (SHAW ments.Intensely hybridizing fragments, including et al. 1984). The membranes were exposed to Kodak XAR- shared fragments that vary ia hybridization intensity 5 film by varying lengths of time at room temperature or at between species, are tabulated for each enzyme and -70" with Dupont Cronex Lightening Plusintensifying screens. each species in Table 2. Analysis of autoradiographs: The approximate size of We conducted a phylogenetic analysis (PAUP, ver- each restriction fragment was ascertained using a custom- sion 2.4, SWOFFORD,1985) of these five Mus species ized computer program written for the Apple Ile and a using the 180 restriction fragments as characters.TWO HIPAD digitizer (Houston Instrument). Phylogenetic anal- minimum length trees were produced from 170 apo- yses using parsimony (PAUP, version 2.4, SWOFFORD1985) were conducted for two sets of taxa using restriction frag- morphic (derived) characters (Figure2). This analysis ments lengths as characters. Fragments of equal molecular substantiates the observed species-specific patterns of weight generated by each restriction enzyme were assumed hybridization because each species branch, including identical between taxa. Each individual taxon was scored M. spretus, M. hortulanus, M. abbotti, M. musculus and for the presence or absence of all restriction fragments M. domesticus, is clearly defined by a series of restric- identified in a given setof taxa. For all phylogenetic analyses, variation incopy number atnong shared restriction frag- tionfragments including the majority of intensely ments was not scored as separate characters and thus the hybridizingfragments. Within species variation of phylogenetic trees produced represent minimal estimates of YB 10 sequences, characterized primarily by faintly the variation among Y chromosomes. In cases where diver- hybridizing restriction fragments, was also observed gent taxa shared fragments of identical size, DNAs from for each species. these taxa were electrophoresed on the same gel to identify The phylogenetic analysis providedan objective more accurately shared fragments. Genomic DNAsfrom all wild caught M. domesticus and M. abbotti were analyzed twice method to infer relatedness among taxa. In both trees and, with two exceptions (Table l), genomic DNAs from a M. musculus and M. domesticus are sister taxa, i.e., they Y Chromosome Evolution in Mus 171
TABLE 1 Species, numberof individuals sampled,original collecting localities and source of taxa used in the analysisof RFLP's in the genus Mus (subgenusMus)
Number Taxa F, M" Original collecting localityb Source' M. spretus 4,4** Spain: Cadiz Prov.; Puerto Real 1 4,4** Morocco: Azrou 1 M. hortulanus 4,4** Austria: Burgenland Prov.; 6 km ENE Halbturn 1 4,4** Yugoslavia: Serbia; 20 km N Pancevo 1 M. abbotti 1,2** Yugoslavia: Macedonia; 6 km NW Gradsko 4 m. musculusdM. m. 3,4** Denmark: Skive 1 4, 6* Denmark: Viborg Co.; Vejrumbro 1 8,8** Czechoslovakia: Moravia; Studenec, approx. 35 km W Brno 1 4, 4* Czechoslovakia: Slovakia; Sladeckovce,approx. 50 km E. Bratislava 1, 3 0, 1 Yugoslavia: Serbia; Belgrade 4 0, 1 Austria: Burgenland Prov.; Halbturn 4 0, I* Austria: Braunau am Inn bezirk; Braunau 4 0, I* Austria: Braunau am Inn bezirk; Ranshofen A. 3 0,1 Austria: Braunau am Inn bezirk; RanshofenB. 4 0, 3* Austria: Braunau am Inn bezirk; Nofing 4 0, 1* F.R.G.: Bavaria; Freising Kreise, Giggenhausen 4 0, 1* F.R.G.: Bavaria; Freising Kreise, Freising 4 0, 2* F.R.G.: Bavaria; Freising Kreise, Achering 4, 3 0, 1* F.R.G.: Bavaria; Freising Kreise, Dornhaselbach 4 0, 8* F.R.G.: Bavaria; Freising Kreise, Rudlfing 4, 3 0,2 F.R.G.: Bavaria; Freising Kreise, Tuntenhausen 4 0,1 F.R.G.: Bavaria; Freising Kreise, Massenhausen 4 0,5 F.R.G.: Bavaria; Erding Kreise, Schwaig 4, 3 0,5* F.R.G.: Bavaria; Erding Kreise, Sonnendorf 4, 3 0, 1* F.R.G.: Bavaria; Erding Kreise, Gut Wildschwaig 4 0,1* F.R.G.: Bavaria; Erding Kreise, Hogersdorf 4 0, 1 F.R.G.: Bavaria; Erding Kreise, Tittenkofen 4 0, 2* F.R.G.: Bavaria; Traunstein Kreise, Traunstein 4 0, 1* F.R.G.: Bavaria; Rottal-Inn Kreise, Mitterskirchen 4 0, 5* F.R.G.: Bavaria; Rottal-Inn Kreise, Simbach 4, 3 m. castaneusM. m. 4,4** Thailand: Chonburi Province, Chonburi 1 m. molossinusM. m. 4,4** Japan: Kyushu 1, 3 0, 1* Japan: Okinawa, Naha 4 M. domesticusd 5, 5* U.S.A.: Texas; College Station 5 U.S.A.: Maryland; Upper Marlboro, University of MarylandTo- bacco Farm 4, 4 Seed shed 1 4, 4 Chemical shed 1 4, 4 Drying barn 1 4,4 Tool shed 1 4,4 USA.: Maryland; Queen Anne Co., Ridgely,J.J. Downs 1 4, 4 U.S.A.: Maryland; Davidsonville, Haven's Farm 1 4,4 U.S.A.: Maryland; Davidsonville, Sanner's Farm 1 8,8** U.S.A.: Maryland; Queen Anne Co., Centreville 1 4, 4 U.S.A.: Delaware; Lewes 1 4, 4* U.S.A.: California; Bouquet Canyon 0, 1" Great Britain: Berkshire; 2 mi SW Abingdon, Culham College 4 0,1 Spain: Barcelona; 0.5 km W San Fausto 4 0, 3* Egypt: Faiyum Governate; AI Faiyum Depression 4 0, 2* Egypt: Giza Governate; near Bashtil 4 0, 1* Israel: Jerusalen 3 0, 2* Italy: lsole Eolie; Lipari 2t 0, 2* Italy: Apennine; Molise 2t 0,2* Italy: Bergamo; Orobie 2t 0, 1* Italy: Laatsch 4 0, 1* Italy: Tuscany; Pisa 4 0, 1* Italy: Sondrio Prov.; Morbegno 4 0,I* Greece: Ellas 3 0,1 Yugoslavia: Dalamatia; Metkovic 4 0,1 F.R.G.: Bavaria; Freising Kreise, Thalhausen 4 0,2 F.R.G.: Bavaria; Freising Kreise, Kammerberg 3 172 P. K. Tucker-, B. K. Lee and E. M. Eicher
TABLE 1 Continued
Sumher -,.. .l\
I‘l(;llRE 1 .-Acltol;ltliogr~~~)l~sof niak genonlic 1)Y.i cut with Pstl ;und probtd with pYBIO. Outside Ianes are Iamhd;r-Hindlll molecular wcighl n~;~rkct.s.hnplrs include the follo\ving species: I;me I = .\I. sprptus. Spin; lane 2 = A!. sprrtus. Morocco; lane 3 = M. hortulanus. /\ustri;l: I;IIIC 4 = .\I. Irortrclanus, Yugoslavia: lane 5 = ,\I. musrulus musrultts. Denmark; lane 6 = M. m. mtcscttlus, CzecI1oslav;lkia; lanes 7 and X = .\I. m. rno1ossinu.r. Japan; lanes $1 ;Ind 10 = .\I. m. rastantus, Thailand: lane 1 I = .\I. domrstirus. Egypt; I;mc I2 = M. domrsticus, U.S.A.; and lanes I3 ;~ndI4 = .\I. ahhotti, Yugoslavia. 1.ow copy nurnher fragments are identified by closed circles. A 2.0-kb Pstl fragment shared anlong a11 tasa, is found in higher copy nunlhcr in ’11. sprttus and .\I. ahhotti. The differcvcc ill intensity of hybridization between the two sample5 of.\l. sprptus is due to an untlerlo;ltling of lane 2. A 2.24)Pstl and ;I 2.6-kt)Pstl fragment shared ;Inlong M. ahhotti, M. musrulus and .\I. rlom~.rtirrrsurics ill copy number actx)ss tasa. Two Pstl high copy ntrmher fr;lgnlents, 3.5-kh and ;I 6.3-kb in length. ;Ire unique to M. domp.rtirfrsand .\I. abbotti respectively. hwcopy nun,ber fragments are either species-specific (the 1.8-kh psll fr;lgment of i\4. dOmPStiCUS) or unique toa populatiotl within ;I species (the 5.7-kl) Pstl fragment of ,\I. dompstirus-U.S.A.). are more closely related to each other than they are ally, M. m.molossinus, M. m. castaneus and M. m. to AI. spretus, AJ. hortulanus and M. abbotti. Addition- musculus are more closely related to each other than Chromosome Evolution in EvolutionY Chromosome Mus 173
TABLE 2 Presence and absenceof restriction fragments found in multiple copies in at least oneof five species in the genusMUS
~~ Species of Mus Mus musculus Fragment Mus Mus Mus Mus Enzyme (kb) spretus hortulanus abbotti musculus castaneus molossinus domesficus Haelll 0.6 - ++ ++ ++ ++ 0.4 + ++ ++ ++ ++ Tag I 5.8 - + + + + 2.8 - + + + - 2.1 + + + + ++ 1.5 ++ + + + +++ 1.3 ++ ++++ ++++ ++++ ++++ Hinfl 2.2 +++ - - - - 1.2 ++ - - - - 1 .0 - ++++ ++++ ++++ ++++ Bgl I I 5.0 +++ ++++ ++++ ++++ ++++ 4.4 - - - - +++ 4.3 +++ - - - - 3.3 - - - - +++ 3.0 - ++ ++ ++ - EcoRI 7.4 + ++++ ++++ ++++ ++++ 6.3 - - - - - 5.4 - ++ - - - 3.7 + ++ + + + 3.2 - - - - - 2.9 + ++ + + + 2.4 - - - - - 1.6 + - - - - 0.9 + - - - - Hind111 2.7 ++ +++ +++ +++ ++ PUUI I 8.9 + +++ +++ +++ +++ 8.4 - +++ +++ +++ +++ 7.7 - - - - ++ 3.8 +++ +" - - - 2.8 - - - - ++ 2.7 - - - - ++b Pstl 6.3 +++ - - - - 3.5 - - - - +++ 2.6 + ++ ++ ++ + 2.2 + +++ +++ +++ ++ 2.0 +++ + + + + The presence or absenceof this fragment varies within the subspecies M. m. musculus. ' The presence or absence of this fragment varies within the species M. domesticus. A [-I in each column indicates the absence of a specific fragment for a given species. A [+I in each column indicates the presence of a fragment. Multiple [+]'s indicate presumedcopy number differencesbased on intensity of hybridization betweenspecies for a given fragment. A greater number of [+]'s indicates the presence of a greater number of copies for a fragment of a specific size. The specific number of [+]'s, however, is not a strict measure of the degree of amplification. Low copy number fragments are notlisted in this table. to M. domesticus, indicating that thesethree subspecies consensus tree (Figure 2C) was constructed from trees comprisea monophyletic group. The relationships A and B. In this tree M. spretus, M. hortulanus, and among M. spretus, M. hortulanus and M. abbotti differ M. abbotti are as distantly related from each other as between the two trees. In tree A (Figure 2A) M. abbotti they are from M. musculus and M. domesticus. and M. hortulanus are more closely related to each To characterize the evolution of YBlO sequences other than they are to M. spretus or to M. musculus further, a more extensive analysis of within species and M. domesticus. In tree B (Figure 2B) M. spretus variation was conducted for M. musculus and M. do- and M. hortulanus are more closely related to each mesticus. Genomic DNA from male mice of 24 geo- other than they are to M. abbotti. When more than graphically isolated populations of M. musculus and one minimum length tree is produced in a pylogenetic 30 geographically isolated populations of M. domesti- analysis a consensus tree can be constructedusing only cus was digested with five restriction enzymes, includ- concordant data from the minimum length trees. A ing TaqI, BglII, EcoRI, PvuII and PstI, and Southern 174 P. K. Tucker, B. K. Lee and E. M. Eicher
18
m. musculus - Denmark M. m. musculus - Czechoslovakia - 5 .* 8 3 M. m. castaneus -Thailand -r(l M. m. molosslnus -Japan -8. 23 .....a 8 M. domesticus - Egypt 8 M. domesticus - U.S.A. o M. hortuknus - Austria 21 - 2 M. hortulanus - Yugoslavia 40 .... ' M. abbotti - Yugoslavia ' M. abbottf - Yugoslavia B 2 M. spretus - Morocco FIGURE3.-An autoradiograph ot male genomlc DNAs troni 18 geographically isolated populations of M. domesticus digested with 10 M. spretus - Spain Tagl and probed with pYBI0. The outside lane is a lambda-Hindlll hortulanus Austria 13 OM. - molecular weight marker. Samples include mice from the following M. hortulanus - Yugoslavia localities: lane 1 = Centreville, U.S.A.; lane 2 = College Station. - U.S.A.; lane 3 = Nurenberg. F.K.G., lane 4 = Heidelberg, F.K.C.; 38 .... 1 M. abbottl - Yugoslavia lane 5 = Ellas, Greece; lane 6 = Zalende. Switzerland (identified as 1 M. abbottl - Yugoslavia M. d. poschiauinus); lane 7 = Iipari, Italy; lane 8 = Orobie, Italy; . musculus - Denmark lane 9 = Molise, Italy; lane 10 = Azrou, Morocco (identified as M. 16 M. m. mUSCulus - Czechoslovakia d. brevirostris): lane 1 I = Erfoud, Morocco (identified as M. d. M. m. cast8neus - Thailand pradexfus); lane 12 = Abu Rawash,Egypt. Mice from Zalende. Orobie, Molise and Lipari are from Robertsonian metacentric - M. m. molosslnus - Japan populations. Low copy number fragments are identified by closed 21 8. M. domesticus - Egypt circles. All M. domesticus sampled share four highcopy number ...... Tag1 fragments that are I .3 kb, 1.5 kb, 2.1 kb and 5.8 kh in length. domestlcus U.S.A. M. - With the exception of a 6.6-kb Tag1 fragment. the variation within C M. domesfirus is characterized by nunlerous low copy number frag- M. spretus ments.
M. spretus blots of these DNAs were probed with pYBlO (Table M. hortulanus 1). In this more extensive second analysis a total of M. hortulanus 185 fragments were identified by single digests of M. m. musculus genomic DNA. The hybridization patterns observed M. m. musculus when male genomic DNA was digested with TaqI (Figure 3) typify thepatterns seen with theother M. m caslaneus restriction enzymes and substantiate the initial obser-
M. m. molossinus vation that within species variation is characterized I primarily by faintly hybridizing restriction fragments. M. domesticus The populations of M. musculus sampled represent three recognized subspecies, 1) two populations of M. 1 M. domesticus m. musculus fromDenmark, two populations from M. 8bbOtti Czechoslovakia, three populations from Austria and eleven populationsfrom The Federal Republic of M. abbottf Germany, 2) one population of M. m. castaneus from FIGURE2.--E\~olutionary trees constructed by a phylogenetic Thailand and 3) two populations of M. m. molossinus analysisusing parsimony (PAUP). Trees A and B represent the most parsimonious trees found from an ;nlalysis of restriction frag- from Japan.The populations of M. domesticus sampled ment length polymorphism data using pYBI0. The trees were included four of the currently recognized subspecies. rooted at the midpoint of greatest patristic distance. The length of each tree is 188 ;~ndthe consistency index, CI (FARRIS1970). is 0.899. The CI. calculated as "the sum of the ranges of a11 characters determine the length of each horizontal branch is given and the in the data divided by the number of evolutionary changes on the number and position of high copy number restriction fragments tree (i.e.. tree length)" (BROOKS, O'GRADYand WIIXY1986). is an along the tree areindicated by asterisks. The phylogenetic relation- indicator of the amount of homoplasy (parallel, reversal, or conver- ships among M. sprdus, M. hortulanus and M. abbotti differ between gent evolutionary events) in a tree. A high CI reflects minimal tree A and tree B. Tree C is a consensus tree constructed using homoplasy. The number of characters (restriction fragments) that only concordant data from trees A and B. Y Chromosome Evolution in Mus 175
However, because the subspecific designations of M. I M. hortulanus domesticus are disputed (FERRISet al. 1983; BON- HOMME et al. 1984), our samples were identified only to species with the exception of M. d. brevirostris from m. musculus M. musculus Morocco, M. d. praetextus from Morocco and M. d. Czrchoslovskls poschiavinus from Switzerland and Italy (Table 1).The remaining samples of M. domesticus arefrom one locality fromGreat Britain, one from Spain, three from Egypt, one from Israel, six from Italy, one from r M. m. csstansua - Thalland Greece, ten from the Federal Republic of Germany and three from the United States. An initial investi- AbuRawa8h. Egypt gation of Y chromosome variation in M. domesticus samples from Delaware and Maryland (Table 1) indi- AI-Falyum, Egypt cated no variation among Y chromosomes of these GIzB, Egypt mice and thus the Y chromosome of M. domesticus Tubingen,F.R.G. from these localities was represented by a single local- ity (Maryland: Queen Anne Co.; Centreville). A subset of the M. musculus and M. domesticus samples reflecting the variation found among Y chro- M. d. bnvlroshls - Azrou,Morocco mosomes was used in a phylogenetic analysis and a series of minimal length trees were produced from MoroccoAzrou, M. domortlcus 117 apomorphiccharacters of the185 characters Jerusalam, Isrssl (restrictionfragments) identified. The most com- monly observed arrangement of taxa among ten trees localltlas In Frslmlng Krelsa, analyzed is represented in Figure 4. The lower con- F.R.G. sistency index for this tree indicates that a large num- ber of characters represented convergent,parallel, or reversal events. These primarily includerestriction fragments found within the M. domesticus group. Pre- sumably, fragments of identical molecular weight, identified as shared fragments, may have evolved in- dependently within the M. domesticus lineage. M. mus- culus and M. domesticus fell into two discrete groups in all trees analyzed indicating that each of these two species comprise a monophyletic group. In addition, Barcelona. Spsln the arrangementof taxa within the M. musculus group 1 Orobls, Italy remained thesame, whereas subgroups of taxa within the M. domesticus group varied in position along the length of the tree in each tree analyzed. In the M. musculus group two distinct groups were found within the subspecies M. m. musculus. These FIGURE4.-An evolutionary tree constructed by a phylogenetic include mice from Denmark and from central Europe. analysis using parsimony. This tree is one in a series of heuristic trees produced from the restriction fragment length polymorphism As in the first phylogenetic analysis (Figure 2), M. m. data using pYB10. Mus hortulanus was used as an outgroup. The molossinus and M. m. castaneus are distinct subgroups tree length is 249. The consistency index is 0.526. The number of within the M. musculus group. In contrast to the first characters (restriction fragments) that determine thelength of each analysis, M. m. molossinus is more closely related to M. horizontal branch is given. m. musculus than is M. m. castaneus. Relationships among mice within the M. domesticus from Egypt; 4) a mouse identified as M. d. praetextus group varied among the treesanalyzed, although cer- from Morocco; and 5) amouse from Maryland (whose tain subgroups of mice were consistently defined by Y chromosome was identical to one of two sampled four or more restriction fragments. Such subgroups from Texas andto the Y chromosome of mice sampled include 1) populations from Orobie,Lipari and Molise from California). Overall, phylogenetic analyses of M. in Italy having Robertsonianmetacentric chromo- domesticus demonstrate the highly variable nature of somes; 2) Robertsonian chromosome bearing popula- the Y chromosome throughout its range. For example, tions from Tirano, Italy, and Zalende, Switzerland, extensive variation exists among Y chromosomes of identified as M. d. poschiavinus; 3) a group of mice M. domesticus from Germany in contrast to the relative 176 P. K. Tucker, B. K. Lee and E. M. Eicher lack of variation observed among M.musculus, in jacent sequences is characterized by bothsequence particular and, among M. m. musculus from Europe. divergence and sequence amplification, whereas in- Finally, a sample of M. musculus and M. domesticus traspecies variation is characterized primarily by se- mice collected along a 160-km transect in the Federal quence divergence. Republic of Germany and Austria (the Austria-F.R.G. We also performed phylogenetic analyses to deter- sample of M. musculus and the Freising-Kreise sample mine whether and atwhat taxonomic level a paternally of M. domesticus) were included in this analysis and inherited repeated DNA sequence contributes toour minimal variation involving one or two fragments was understanding of the phylogenetic relationships in the identified among Y chromosomes within either spe- genus Mus. Ostensibly, the useof a multiple copy cies. DNA sequence for phylogenetic analyses is problem- atic. Restriction fragments of arepeated sequence DISCUSSION cannot be mapped to specific sites and thus it is impossible to ascertain whether the presence or ab- EICHERet al. (1989) have suggested that YBlO sence of each restriction fragment represents an in- sequences arose on the Y chromosome of the ancestor dependent evolutionary event. For example, different common to M. spretus, M. abbotti, M. hortulanus, M. size restrictionfragments can be produced by site musculus and M. domesticus as these are theonly species changes or by amplification events. In the latter case, in the subgenus Mus that have YB 10 sequences exclu- different size fragments generatedby different restric- sively on the Y chromosome. They also suggested that tion enzymes may actually reflect only a single ampli- the species-specific patterns of hybridization observed fication event. with YBlO resulted from both sequence divergence In spite of this problem, there is strong evidence to and sequence amplification of the Y chromosome dur- suggest that the YBlO repeat is phylogenetically in- ingsubsequent speciation events. In this paper we formative. This is evidenced from the similarities be- have further investigated the evolution of YBlO and tween the phylogenetic trees depicted in Figure 2 and adjacent Y chromosome sequences in these five species phylogenies of the same taxa constructed from a va- at both the species and population levels. We found riety of data sets. For example, aconsensus of the two that Y chromosome differences between species are trees depicted in Figure 2 is concordant with trees characterized by variation in both intensely and faintly generated from mitochondrialDNA data of FERRISet hybridizing fragments whereas Y chromosome differ- al. (1983) with regard to the position of M. spretus, ences within species are characterized primarily by M. hortulanus and M. abbotti. In both the mitochon- variation in faintly hybridizing fragments. drial DNA and Y chromosome analyses these taxa are Variation in hybridization intensity of YBlOse- quences could be due to either differences incopy as different from each other as they are from the M. musculus/M. domesticus lineage. This is in contrast to number or todifferences in sequence homology. How- ever, we interpret the observed differencesin intensity phylogenetic trees generated from biochemical data of hybridization among restriction fragments as pri- that place M. hortulanus and M. abbotti as sister taxa marily reflecting variation in copy number of YBlO (BONHOMMEet al. 1984) and aphylogenetic tree gen- sequences, intensely hybridizing fragments being high eratedfrom differences in the 5’ end of the16s copy number fragments and faintly hybridizing frag- mitochondrial rDNA molecule (FORT et al. 1984; ments being low copy number fragments. The reason BONHOMME1986) that places M.hortulanus (= M. for this interpretation results from two observations. spicilegus) as a sister taxa tothe M. domesticuslM. No new fragments were observed when M. spretus, M. musculus lineage. The position of M. m.castaneus hortulanus, M. abbotti, M. musculus and M. domesticus within the M. musculus cluster in all the phylogenetic DNAs were hybridized with pYBlO at lower strin- trees (Figures 2, A and B, and 4) contrasts with its gency conditions and hybridization intensity increased placement as a distinct lineage in phylogenetic trees for all restriction fragments across taxa following a generated from the mitochondrialDNA (FERRISet al. decrease in stringency conditions (EICHERet al. 1989). 1983)but its position in the second phylogenetic We, thus, interpret variation in intensity of hybridi- analysis (Figure 4) is concordant with the phylogenetic zation among equal sized (shared) fragmentsas reflect- relationships generatedfrom biochemical data of ing amplification of YBlOsequences. Additionally, we BONHOMMEet al. (1984). In addition, the placement suggest that presumed high copy number fragments of M, m, musculus and M. m. molossinus as sister taxa thatare unique to a given taxon reflect sequence relative to M. m. castaneus and M. domesticus in the divergence and amplification of Y chromosome se- second phylogenetic analysis (Figure 4) isin agree- quences whereas presumed low copy fragments ment with phylogenetic analyses generated from mi- unique to a given taxon reflect sequence divergence tochondrial DNA data (FERRISet al. 1983; YONEGAWA such as single restriction site changes. In general, we et al. 1986), biochemical data (BONHOMMEet al. 1984), conclude that interspecies variation of YB 10 and ad- nontranscribed spacer regions of ribosomal DNA (SU- Y Chromosome Evolution in Mus 177
ZUKI et al. 1986), T-lymphocyte differentiation anti- some populations from Orobie, Molise and Lipari is gen types (KURIHARAet al. 1985; MORIWAKIet al. surprisingfor two reasons. These populations are 1986) and &-microglobulin types of the class I major geographically isolated from each other and,with the histocompatibility antigens (ROBINSONet al. 1984). exception of two Robertsonian chromosomes shared The placement of M. musculus and M. domesticus as between theOrobie and Molise populations, each sister taxa relative to M. spretus, M. hortulanusand M. carries a different arrangementof Robertsonian chro- abbotti agrees with phylogenetic trees generated from mosomes (reviewed in SAGE198 1). The similarity of boththe mitochondrial and biochemical data sets. YB 10 sequences among these populations suggests Finally, the position of M. domesticus and M. musculus that they have a common origin. as monophyletic taxa is in accordance with all the data The inability to distinguish mice identified as M. d. sets in the above comparisons. We conclude that the domesticus or M.d. brevirostris using the YBlO se- similarities among the phylogenetic relationships gen- quence corroborates the findings of the biochemical erated by the Y chromosome data and other data sets and mitochondrial DNA data (SAGE1978, 1981;BON- indicate thatthe paternallyinherited repeated se- HOMME et al. 1978; BRITTONand THALER1978; FER- quence YBlO provides phylogenetic information RIS et al. 1983). Although the biochemical and mito- within and among a closely related group of species. chondrial data suggest otherwise, our single sample Inconsistencies among the trees generated by differ- identified as M. d. praetextus is clearly distinguishable ent sets of characters probably reflect the close evo- on the basis of its Y chromosome type from other M. lutionary relationships of these taxa. domesticus and may reflect a real subspecific differ- Variation of Y chromosomes among M. spretus, M. ence. Additionally, a M. domesticus Y chromosome type hortulanus, M. abbotti, M. musculus and M. domesticus identified in North America is distinct from the Y is indicative of evolution of this Y chromosome re- chromosome types sampled in Europe and the Medi- peated sequence within the last 3-5 million years as terranean region.Presumably, this Y chromosome M. spretus, M. hortulanus and M. abbotti are hypothe- type exists in an, as yet, unidentified population of M. sized to havediverged from M. domesticus and M. domesticus in Europe, assuming that the presence of musculus 3-5 million years ago (FERRISet al. 1983). M. domesticus in N. America is associated with Euro- Variation of Y chromosomes within the M. domesticus pean colonization. and M. musculus samples is indicative of a rapid rate Documentation of the rapid rate of evolution of a of evolution of this Y chromosome repeated sequence. Y chromosome specific repeated DNA sequence in a If M. musculusand M. domesticus have diverged within group of closely related species presents many intrigu- the last 1-2 million years (FERRISet al. 1983), Y ing questions. For example, does a DNA sequence chromosome change has taken place within both lin- have aunique mode of evolution by virtue of its eages at the population level since that time. isolation on a monosomic chromosome? The exclusive Greater within species variation among Y chromo- occurrence of YB 10 sequences on the Y chromosome somes in the M. domesticus samples compared to the in the subgenus Mus and its restriction onthe Y M. musculus samples can be attributed to either dis- chromosome to a region that does not undergo recom- parities in sampling, as the major portion of our M. bination with the X chromosome (EICHER,PHILLIPS musculus samples are from central Europe and thus and WASHBURN1983) precludes mechanisms of am- may represent a single migration of this species into plification that involve homologous (or nonhomolo- that region from central Asia, or to differential mu- gous) chromosomeexchange. Amplification of this tation or fixation rates between these two species. It sequence could occur by unequal exchange between is interesting to note that most of the polymorphism sister chromatids of the Y chromosome or by a process for the t-complex of genes in European populations of duplicative transposition (ORGEL andCRICK 1980; of wild house mice is found within M. domesticus and DOOLITTLEand SAPIENZA1980) limited in this in- not within M. musculus (KLEIN, SIPOSand FIGUEROA stance to theY chromosome. Are otherY chromosome 1984). This suggests that the evolution of the t-com- specific DNA sequences evolving in a like manner, plex and theY chromosome in these two species reflect i.e., is sequence amplification and divergence a general common histories. phenomenon of the mammalian Y chromosome? The divergent Y chromosomes of the Robertsonian Finally, a rapidly evolving paternally inherited DNA metacentric chromosome populationsof M. domesticus sequence has contributed in a novel way to under- sampled (all withgreater thanfive Robertsonians) may standing the phylogenetic relationships in a closely reflect the reproductive isolation of these Robertson- related group of species in the subgenus Mus. More ian populations from surrounding M. domesticus with extensive studies of Y chromosome variation within the normal (2N = 40) acrocentric karyotype (CA- species also could provide important information on PANNA et al. 1976). The similarity among the Y chro- population structure, including identification of foun- mosomes of the Robertsonian metacentric chromo- der events and bottlenecks, in much the same way as 178 P. K. Tucker, B. K. Lee and E. M. Eicher maternally inheritedmitochondrial DNA. Con- FERRIS,S. D., R.D. SAGE,E. M. PRAGER,U. RITTE and A. 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