Tracing Paternal Ancestry in Mice, Using the Y ... - Semantic Scholar

Tracing Paternal Ancestry in Mice, Using the Y-linked, Sex-determining Locus, Suy Barbara L. Lundrigan and Priscilla K. Tucker Museum

of Zoology

and Department

of Biology, University

of Michigan

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y-linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 5 15-bp region from the male sex-determining locus, S-y, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera-Mustomys and Hylomyscus. Percent sequence divergence was ~0.0 1% for comparisons between populations within a species and was 0.19% 8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single mostparsimonious tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports ( 1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (A4. musculus, A4. domesticus, M. spicilegus, A4. macedonicus, and M. spretus) and an Oriental group (M. cookii, A4. cervicolor, and M. caroli), and ( 3) sistergroup relationships between M. spicilegus and A4. macedonicus and between M. cookii and M. cervicolor. We argue that Y-chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.

Introduction

The Y chromosome is unique among chromosomes in the mammalian genome, because only a small portion of it recombines during meiosis; the majority of the Y chromosome is clonally inherited from father to son. One consequence of this mode of inheritance is a potential for providing phylogenetic information through the tracing of paternal lineages. Because nonrecombining Y sequences are paternally inherited, they represent a distinct new class of phylogenetic characters to complement maternally inherited mitochondrial DNA (mtDNA) and Mendelian inherited characters. In addition, like mtDNA sequences, these clonally inherited sequences contain a historical account of molecular evolution that is not confounded by the effects of recombination. Y-linked sequences might therefore make especially useful markers for investigating historical relationships among populations within species and for tracing male-mediated gene flow. Despite this potential, there have been no published phylogenies based on Y-chromosome sequence data. Key words: Y chromosome, Sry, molecular systematics, MUX Address for correspondence and reprints: Barbara L. Lundrigan, Museum of Zoology, University of Michigan, Ann Arbor, Michigan 48 109- 1079. Mol. Biol. Evol. 11(3):483-492. 1994. 0 1994 by The University of Chicago. All rights reserved. 0731-4038/94f I103-0015$02.00

This is primarily because such studies depend on finding a region of the Y chromosome that is found only on the Y chromosome and that is variable at the appropriate level among the taxa of interest. The few phylogenetic studies that have utilized the Y chromosome are based not on sequence data, but on Southern blot hybridizations in which sequences that are unique to the Y chromosome of humans or laboratory mice were used as hybridization probes to Southern blots of enzyme-digested genomic DNA from related taxa. The resulting hybridization patterns suggest that sequences that are unique to the Y chromosome across a broad range of taxa are rare. Most human sequences that are unique to the Y chromosome are not found on the Y chromosome in other primates (Cooke et al. 1982; Kunkel and Smith 1982; Page et al. 1984; Koenig et al. 1985; Erickson 1987)) and most laboratory mouse sequences that are unique to the Y chromosome are conserved on the Y chromosome only among closely related taxa within the subgenus Mus (Nishioka and Lamothe 1986; Platt and Dewey 1987; Either et al. 1989; Tucker et al. 1989). However, the Y chromosome does include some recently described functional loci, including Zfv (Page et al. 1987), Sry (Gubbay et al. 1990; Sinclair et al. 1990)) and Ubely (Mitchell et al. 199 1) , that are unique to the Y chromosome across a broad range of mammalian species. Both Zfv and Ubely have “ancestral” 483

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homologues on the X chromosome (discussed in Tucker et al. 1992b). The purpose of this study is to investigate the use of Y-chromosome sequences for phylogenetic inference by examining sequence variation at the male sex-determining locus, Sry (Gubbay et al. 1990; Sinclair et al. 1990). We describe a comparative analysis of a 5 15bp region from Sry in 22 representatives from the rodent subfamily Murinae, family Muridae. Variable nucleotide sites from the murine Sry homologues are used as characters in a phylogenetic analysis using parsimony. The resulting phylogeny is compared with phylogenies constructed using maternally inherited mtDNA and Mendelian inherited characters (data originally published in She et al. [ 19901 and reanalyzed in the present study). Material and Methods DNA Samples and Sample

Used in the Investigation of Sry Evolution

SrY

Collecting Locality

Taxon a Genus Mus subgenus Mus Mus musculus musculusb

castaneus

.

Mus domesticus (2n = 26)

(2n = 24) (2n = 40)

Preparation

The genus Mus, as currently defined using a Linnean classification, encompasses 30-40 species in the following four subgenera: Mus, Coelomys, Pyromys, and Nannomys (Marshall 1977, 198 1). In the present study, we sampled 20 individuals from the genus Mus, including eight species from the subgenus Mus and one from the subgenus Coelomys (Mus pahari) . We also sampled representatives from two additional murine genera, Mastomys and Hylomyscus. Original collecting localities for wild mice obtained from laboratory colonies and for field caught mice are given in table 1. Genomic DNA was extracted from frozen tissue according to the methods of Jenkins et al. ( 1982).

(2n = 40)

...

(2n = 22)

...

(2n = 40)

...

..

Mus spicilegus

Mus macedonicus . . . . . . . . Mus spretus . . . . . . . . . . . . . .

...... ....

Mus cookii

Reaction

(PCR)

Amplification

Mus caroli

.

..

..

Mus cervicolor popaeus Mus cervicolor cervicolor

Sry was cloned from the male sex-determining region of an inbred mouse Y chromosome (Gubbay et al. 1990). It is situated within 2.8 kb of unique sequence surrounded by a long, inverted duplication (Gubbay et al. 1992 ) . The transcript is thought to be composed of a single exon with an open reading frame consisting of a highly conserved 79-amino-acid high-mobility group DNA binding domain (HMG box) flanked by an Nterminal region of 2 amino acids and a C-terminal region of 3 14 amino acids (A. Hacker and R. Lovell-Badge, unpublished data). Polymerase Chain DNA Sequencing

Table 1 Specimens

.

. ..

Japan: Kyushu Denmark: Viborg Country, Skive Czechoslovakia: Slovakia; Sladeckovce Thailand: Chonburi Province; Chonburi Switzerland: Grisons Canton; Zalende, Poschiavo Italy: Sondrio; Tirano Morocco: Tafilalt Oasis; Erfoud United States: Maryland; Queen Anne’s County, Centreville Italy: Molise; 6.1 km W by road of Bonefro Italy: Lazio; 11.4 km WNW by road of Cassino train station Austria: Burgenland Province; 6 km ENE Halbturn Yugoslavia: Debeljaca Yugoslavia: Gradsko Morocco: Azrou Spain: 8 km E Puerto Real Thailand: Tak province; Loei Thailand: Saraburi Province Thailand: Chonburi Province Thailand: Chonburi Province

Subgenus Coelomys Mus pahari

...

... ..

Thailand: Tak province

.. ...

Kenya: Eastern Province; Machakos District Gabon: Estuaire Province; 1 km southeast of Cape Esterias

Outgroup taxa: Mastomys hildebrantii Hylomyscus alleni

.

and

A 47 I-bp region of S-y (8207-8677; Gubbay et al. 1992), beginning 103 bp 5’ to the HMG box, was enzymatically amplified from genomic DNA (Saiki et al. 1985) by using two oligonucleotide primers-5 ‘AGATCTTGATTTTTAGTGTTC-3 ’ and 5 ‘-GAGTACAGGTGTGCAGCTCTA-3 ‘. Two additional

primers, 5’-TCCTACACAGAGAGAAATACC-3 ’ and 5 ‘-CACCAGTGATGTCAGCTGTTAGTA-3 ’ ( spanning 849 l-9435 in the inbred mouse; Gubbay et al. 1992), were used to amplify a region of variable length that overlaps for 187 bn with the 3’ end of the 47 1-bp

Tracing Paternal Ancestry

fragment. The amplification protocol included 25 cycles, with denaturation at 95°C for 1 min, annealing at 50°C for 1 min, and elongation at 72°C for 1 min 15 s. To verify that amplified sequences were unique to the Y chromosome, preliminary amplification experiments included both male and female genomic DNA from each species under investigation. Amplification of the target size sequence in males only was taken as evidence that the sequence was unique to the Y chromosome. DNA was sequenced using the Sanger dideoxy-sequencing technique (Sanger et al. 1977). In our initial experiments, single-stranded DNA was generated from the double-stranded product and was sequenced using the Sequenase version 2.0 sequencing kit (United States Biochemical). Subsequently, we used the Cycle Sequencing System (GIBCO BRL). Sequence Alignment Sequences were aligned with the published sequence from inbred mouse strain 129 (Gubbay et al. 1990) by using the SS2 algorithm (Altschul and Ericson 1986), available in the Eugene package of sequence analysis programs. The 3’ end of the second amplification product included a CAG repeat of variable length, which could not be unambiguously aligned (Tucker and Lundrigan 1993); we excluded that region from the present study. Phylogenetic Analyses Aligned sequences were subjected to parsimony analysis using PAUP, version 3.1.1 ( Swofford 1993 ) . Characters were unordered ( Fitch optimization ) and uniformly weighted. We performed a single analysis, using the branch-and-bound option, with Mastomys hildebrantii and Hylomyscus alleni as outgroups. These African species represent 2 of 122 genera currently included with Mus in the subfamily Murinae (Musser and Carleton 1993). The historical relationships among murine genera are poorly understood. A decay index ( “Bremer support,” Bremer [ 1988 ] ; and “decay index,” Kallersjo et al. [ 1992 ] ) was calculated for each clade in each tree topology using a program written by D. Eernisse (Eernisse and Kluge 1993 ) . The decay index of a given clade is the number of additional steps required to generate a nonminimal length tree in which that clade has been dissolved. To compare our phylogenetic reconstruction based on Y-chromosome sequences with phylogenies based on maternally inherited and Mendelian inherited characters, we conducted three additional parsimony analyses by using the following character sets: ( 1) presence or absence of 77 mapped mtDNA restriction sites in eight taxa (data from She et al. 1990)) (2) electromorphs ob-

485

served at 28 protein loci in 10 taxa (data from She et al. 1990)) and (3) the combined data (total evidence) from Sry, mtDNA, and allozymes. Since there was no appropriate outgroup in the mtDNA data set, we used midpoint rooting for this analysis. Mastomys erythroleucus was used as an outgroup for the analysis of the allozyme data. For the total evidence data analysis, we used Mastomys (a composite of Mastomys hildibrantii and Mastomys erythroleucus) and Hylomyscus alleni as outgroups. Character Incongruence To compare the S-y, mtDNA, and allozyme data sets, we used the approach outlined by Kluge ( 1989), which employs the Mickevich-Farris character incongruence metric (Mickevich and Farris 198 1). Cladograms were constructed from each set of characters and from the combined characters and were compared with respect to the number of extra steps (i.e., the minimum number of steps needed to simultaneously explain all the data on the cladogram minus the minimum number of steps needed to explain the data when each character’s hypothesized history is considered separately). The number of extra steps in each of the Sky, mtDNA, and allozyme trees was subtracted from the number of extra steps in the total evidence tree, to give the number of extra steps in the total evidence tree resulting from incongruence between data sets. This number, divided by the number of extra steps in the total evidence tree, is the character incongruence metric, i.e., the proportion of character incongruence that results from combining data sets (see Mickevich and Farris 198 1) . Results

The target sequences were amplified from male genomic DNA of each of the rodent taxa examined in this study. No amplification resulted when female genomic DNA was used as a template, in accord with the hypothesis that Sry is unique to the Y chromosome. Intraspecific Sequence Divergence An alignment of the variable sites from a 5 15-bp region of S-y is presented in figure 1. Our Asian Mus musculus musculus sequence was identical to that of inbred mouse strain 129 (Mmu 1) but differed from the other representatives of M. musculus, i.e., M. musculus castaneus and European M. musculus musculus (Mmu 2), at two nucleotide sites. This finding is consistent with the hypothesis that the M. musculus-type Y chromosome of the “old” inbred strains of mice is of Asian origin (Tucker et al. 1992a). We found no intraspecific variation in M. domesticus ( n=6 ) , M. spicilegus ( n=2), or M. spretus ( n=2) and only a single-base-pair substi-

486

Lundrigan

Mmu (1) Mmu (2)

and Tucker 888888888888888888888888888888888888888888888888888888888888888888888888888888 2222222222222233333344444444445555555555555555566666666666666666666666677777777

888888 777777

445556667777881246791224557891333455666777777900001223334455566777778800000111 370140452378456445507359064416579203156035789436785380172515616124781712456134

112233 677912

tattaaaggtgggtGCTACCGAGCCAGTTCCGGcTCTATAGcGC~CTCTGACTGACGCAGGATGACCAGGGAGCTAT.TCCCTG __________-------___-_______ C------------------------------_______----~----C_-

. ------

FIG. 1.-Variable nucleotide sites from a 5 15-bp region of Sry, for 12 murine rodents. All sequences are aligned to inbred mouse strain 129 ( Mmu 1; Gubbay et al. 1992), which has the same sequence as Asian Mus rnusculus musculus (see table 1). Numbers in vertical columns correspond to the nucleotide positions in Gubbay et al. ( 1992). The 5’ untranslated region is represented by lowercase letters, and the translated region, by capital letters. Dashes indicate sequence identity; and letters indicate nucleotide substitutions. There is a single-base-pair insertion between positions 87 15 and 87 16 in Mhi and Hal. Mmu ( 2) = Mus musculus castaneus and European Mus musculus musculus; Mdo = Mus domesticus; Mspi = Mus spicilegus; Mma = Mus macedonicus; Mspr = Mus spretus; Mco = Mus cookii; Mce = Mus cervicolor; Mea = Mus caroli; Mpa = Mus pahari; Mhi = Mastomys hildibrantii; Hal = Hylomyscus alleni.

tution (an autapomorphy ) distinguishing A4. cervicolor popaeus from iW. cervicolor cervicolor; thus, each of these taxa is represented in figure 1 by a single individual. Interspecific

Sequence

Divergence

Interspecific sequence divergence ranged from 0.19% (between M. spicilegus and M. macedonicus) to 8.16% (between Hylomyscus alleni and Asian M. musculus musculus and between Hylomyscus alleni and M. cookii; table 2 ) . The nucleotide substitution frequency was relatively low within the 237-bp HMG box but increased markedly immediately 3 ’ to the HMG box. Transition-to-Transversion sions

quence divergence (table 2). A marked excess of transitions relative to transversions, followed by a decrease in this ratio (because of multiple transition substitutions), is the expected pattern when increasingly more divergent taxa are compared (Brown et al. 1982). The absence of a decrease in the transition-to-transversion ratio at the higher levels of sequence divergence suggests that these taxa have not diverged sufficiently for multiple substitutions to be common, thus differential weighting of transitions and transversions in the phylogenetic analysis was not warranted. Phylogenetic

Ratio

The relative frequency of transitions to transverwas 0.50-4.00 and tended to increase with se-

Analyses

Our phylogenetic analysis based on sequence ation in Sry resulted in a single most-parsimonious

varitree,

Table 2 Pairwise Percent Sequence Divergence (Substitutions per 100 Sites above the Diagonal), Calculated by the Jukes and Cantor Method (1969), and Transition to Transversion Ratio (below the Diagonal) for a 515-bp Region of Sry Mmu (1) Mmu(l) .. Mmu (2) . . Mdo . Mspi .. . Mma . Mspr . . Mco . . . . . Mce . . . . . Mea . . . . . . Mpa ... Mhi . . . . . Hal . . . .

NA 3.00 1.33 1.67 3.50 1.86 3.40 3.75 2.75 2.60 3.20

Mmu (2)

Mdo

Mspi

Mma

Mspr

Mco

Mce

Mea

Mpa

Mhi

Hal

0.39

0.78 0.39

1.36 0.97 0.58

1.55 1.17 0.78 0.19

1.75 1.36 0.97 0.78 0.97

4.08 3.69 3.30 3.88 4.08 4.27

4.27 3.88 3.50 4.08 4.27 4.47 2.52

3.69 3.30 2.91 3.50 3.69 3.88 2.33 2.14

5.83 5.63 5.24 5.83 6.02 6.21 5.44 5.24 4.66

6.99 6.80 6.41 6.99 7.18 7.38 6.21 6.02 5.44 5.83

8.16 7.96 7.57 7.57 7.77 7.96 8.16 7.96 7.38 6.99 4.65

1.oo 0.67 1.00 2.50 1.57 3.00 3.25 2.63 2.50 3.10

0.50 1.00 4.00 1.67 3.50 4.00 2.86 2.67 3.33

NA 3.00 1.38 2.50 2.60 2.33 2.27 2.55

4.00 1.50 2.67 2.80 2.44 2.36 2.64

2.00 3.60 4.00 3.00 2.80 3.10

2.00 1.20 1.89 1.82 2.73

2.67 2.86 2.44 3.56

3.00 2.50 3.75

2.75 3.50

3.00

NOTE.-Mmu (I) = inbred mouse strain 129; Mmu (II) = A4us musculus castaneus and European A4us musculus musculus; Mdo = Mus domesticus; Mspi = Mus spicilegus; Mma = Mus macedonicus; Mspr = Mus spretus; Mco = Mus cookii; Mce = Mus cervicolor; Mea = Mus caroli; Mpa = Mus pahari; Mhi = Mastomys hildibrantii; and Hal = Hylomyscus alleni.


with 92 steps, and a consistency index, excluding uninformative characters, of .88 1 (fig. 2 A and table 3). In this phylogeny, the subgenus Mus, as represented by the eight species considered here, is monophyletic. Within the subgenus Mus, the five Palearctic species (M. musculus, M. domesticus, M. spicilegus, M. macedonicus,

A.

I-(2)

7 I-

487

and AL spretus) form a clade separate from the strictly Oriental species (M. cookii, hf. cervicolor, and M. caroli). (The former species are Palearctic in distribution, except M. musculus, which extends into the Orient.) Although there is resolution within these clades (i.e., M. spicilegus and M. macedonicus are more closely related to each

r

M. m. musculus (1)

M. m. musculus (2)

M. m. musculus (2) M. domesticus M. domesticus -

M. spicilegus

M. spretus

M. macedonicus M. spicilegus -

M. spretus M. cookii

M. macedonicus

M. cervicolor M. cookii M. caroli M. pahari

M. cervicolor

M. caroli

-

C.

M. m. musculus (2)

D.

M. m. musculus (1) M. m. musculus (2)

M. domesticus -

M. domesticus

M. spicilegus M. spicilegus 2

M. macedonicus

0

M. macedonicus M. spretus

I

A

M. spretus M. cookii

M. cookii 15 M. cervicolor

M. cervicolor

lo -

h

M. caroli

M. caroli M. pahari M. pahari

MASTQMYS

FIG. 2.-Results of parsimony analyses, performed using the branch-and-bound option in PAUP (version 3.1.1), with unordered, uniformly weighted characters. Outgroup taxa are in capital letters. A, Single most-parsimonious tree, derived from an analysis of nucleotide sequence variation in a 5 15-bp region of Sry. B,Single most-parsimonious tree, derived from an analysis of the presence or absence of 77 mapped mtDNA restriction sites and midpoint rooted (data from She et al. 1990). C, Strict consensus of eight most-parsimonious trees, derived from an analysis of electromorphs observed at 28 protein loci (data from She et al. 1990). D, Strict consensus of two most-parsimonious trees derived from an analysis of the combined data from By, mtDNA, and allozymes. The number of unique and unreversed synapomorphies is given above the branch, and the decay index, in parentheses, below the branch for each nonterminal clade. Summary statistics are given in table 3. M. m. musculus( 1) = Asian Mus musculus musculus; M. m. musculus( 2) = M. musculus castaneus and European M. musculus musculus.

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Lundrigan

and Tucker

Table 3 Summary of Phylogenetic Analyses based on Svy, mtDNA, Allozymes, and the Combined Data Sets

W No.oftaxa ...................... No. of informative characters .... Minimum no. of synapomorphies No. of equally parsimonious cladograms . ........ . . . Length of most-parsimonious cladogram .. .. ....... No. of extra steps . . . . . . . . . Consistency index, excluding uniformative characters .. .. Source.-Mitochondxial

Allozymes

Combined

12 35 87

8 38 70

10 16 90

12 89 247

1

1

8

2

92

5

99 29

99 9

292 45

.881

567

.650

.662

DNA and allozyme data were taken from She et al. 1990 and were reanalyzed.

other than either is to A4. spretus; and A4. cookii and M. cervicolor are more closely related to each other than either is to M. caroli), these groupings are only weakly supported. The topology of the mtDNA tree (fig. 2 B and table 3) is concordant with the tree based on Y-chromosome sequence data, except in the placement of M. spretus, which in this tree is the sister group to M. musculus and M. domesticus rather than to M. spicilegus and M. macedonicus. The tree is fully resolved; however, of the three data sets, the mtDNA data set has the highest level of homoplasy, with 24 of the 38 informative characters homoplasious on the most-parsimonious tree. An analysis of the allozyme data resulted in eight most-parsimonious trees (table 3 ) . A strict consensus is shown in figure 2C. This tree supports the monophyly of the subgenus Mus and the association of the strictly Oriental taxa (M. cookii, M. cervicolor, and M. caroli) but provides no additional resolution. The two equally most-parsimonious cladograms from an analysis of the total evidence (table 3) are represented as a strict consensus cladogram in figure 2D. The topology of this tree differs from that of the Sry tree only in the placement of M. domesticus and A4. spretus. In the total evidence tree, M. domesticus and M. musculus are sister taxa, and M. spretus is part of an unresolved trichotomy. The total evidence tree is more robust than the trees that are based on any data set taken alone, with only the clade containing the two M. musculus types and the clade containing the strictly Oriental taxa supported by fewer than four unique and unreversed synapomorphies. Character

mtDNA

Incongruence

A total of 43 extra steps explain the character incongruence in the separate data sets: 5 extra steps were

required to explain the Sry data on the best-fitting cladogram extracted from that data set, 29 extra steps from the mtDNA data set, and 9 extra steps from the allozyme data set (table 3 ) . The total evidence data set had 45 extra steps, which leaves 2 steps originating from incongruence between data sets. Thus, only 4.4% (2/ 45 ) of the total character incongruence in the combined analysis is due to incongruence between data sets. Discussion

Rate of Sequence Interspecific

Divergence

sequence divergence in this portion of to, or somewhat greater than, that for many functional autosomal genes (Tucker and Lundrigan 1993). However, we found surprisingly little sequence divergence among populations within species. This pattern might reflect the small effective population size of Y-linked genes, compared with autosomal and X-linked genes: when the male-to-female breeding sex ratio is 1, Y-linked genes are only one-quarter as numerous as autosomes and one-third as numerous as X chromosomes (Rice 1988). In addition, there is some evidence that species within the genus A4us are polygynous ( Singleton and Hay 1982) ; this would reduce even further the effective population size of Y chromosomes. Alternatively, intraspecific monomorphism for this portion of S-y might result from genetic hitchhiking, in which a selectively favored mutation sweeps through all populations of a species, fixing alleles at linked loci (Maynard Smith and Haigh 1974; Rice 1987; Kaplan et al. 1989). The extent of the genetic hitchhiking effect is negatively correlated with the amount of recombination in a region (Begun and Aquadro 1992). A selective sweep on the nonrecombining portion of the Y chromosome could result in fixation of almost the entire Y chromosome.

Sry is comparable


The low level of polymorphism in this portion of Sry virtually precludes its use for investigating historical relationships among populations within species, a task for which mtDNA has proved especially useful. In the present study, we were able to detect a probable founder event (i.e., the origin of the inbred M. musculus-type Y chromosome from Asian M. musculus musculus); however, in light of the generally low level of polymorphism exhibited by Srv, much longer DNA sequences would typically be needed, to obtain useful information about intraspecific gene flow in these rodents. Probes consisting of Y-enriched or Y-specific DNA repeat sequences have revealed restriction fragment length polymorphisms among human (Casanova et al. 1985) and house-mouse (M. domesticus) Y chromosomes (Tucker et al. 1989), respectively. Although these polymorphisms can be used for inferring evolutionary relationships, they are problematic, because restriction fragments of repeat sequences cannot be mapped to specific sites. We do not know whether the extremely low level of polymorphism exhibited by Sry is typical for genes located on the clonally inherited portion of the Y chromosome. In general, it has proved difficult to model the evolution of Y-linked sequences because of the many, often conflicting, factors that contribute to the rate and pattern of nucleotide substitution on this chromosome (e.g., see Charlesworth et al. 1987; Miyata et al. 1987; Rice 1988 ) . Additional empirical data are needed to test the generality of this result. Comparison of Data Sets We found almost perfect concordance between our tree based on Srv data and trees constructed using mtDNA and allozyme data. The only inconsistency among the three tree topologies was in the placement of M. spretus, which is the sister group to A4. spicilegus and M. macedonicus on the S-y tree and to M. musculus and M. domesticus on the mtDNA tree. This discrepancy may reflect the low number of informative characters available for comparisons within the subgenus Mus, particularly in the S-y data set, where interspecific sequence divergence within the Palearctic group was < 1.75%. Although character congruence between data sets was extremely high in this study, it is easy to imagine instances where this would not be the case. Y-linked sequences, mtDNA sequences, and autosomal sequences have different transmission properties, and both lineage sorting and differential introgression can result in data sets that are internally consistent but yield tree topologies that are markedly different from one another. Incongruence due to lineage sorting is most likely to occur in comparisons among recently diverged taxa,

489

which retain ancestral polymorphisms in Y-linked or mitochondrial sequences, causing them to appear paraphyletic or polyphyletic with respect to those characters (see Neigel and Avise 1986; Avise et al. 1987). The low level of polymorphism in our Sry data makes lineage sorting in the Sry tree unlikely. However, higher levels of polymorphism may have existed in ancestral populations, and, if species in the genus Mus diverged within the past 7 Myr, as the fossil data suggest (Jacobs et al. 1990), then ancestral polymorphisms would have a high probability of surviving in extant lineages. A number of studies have examined the introgression of mtDNA and autosomal genes across a species boundary (e.g., mice, Ferris et al. [ 19831; Drosophila, Powell [1983]; deer, Car-r et al. [1986] and Cat-r and Hughes [ 1993 ] ; and voles, Tegelstrom [ 19871) . In most cases, introgression was found to be differential, such that the mitochondrial genome was able to cross the species boundary, while the nuclear genome was not ( reviewed in Aubert and Solignac 1990). This can result in individuals bearing the mtDNA of one species and the nuclear DNA of another. In studies of the movement of Y-linked loci across the hybrid zone between M. musculus and M. domesticus in central and southern Europe, no introgression was detected (Vanlerberghe et al. 1986, 1988; Tucker et al. 1992 c) . The failure of Y-linked sequences to cross this hybrid zone is thought to result from either limited successful migration of males or an incompatibility between Y-linked genes and a hybrid genetic background. Laboratory studies of the genetic basis of species differences in a number of taxa have shown that the X and Y chromosomes play a key role in hybrid sterility and inviability (reviewed in Coyne and OK 1989). In the majority of documented interspecific hybridizations, the Fl heterogametic sex is sterile or inviable, while the homogametic sex is fertile (Haldane’s rule; Haldane 1922). If mammalian Y-linked loci typically interact with autosomal or X-linked loci in some critical speciesspecific way, then introgression of Y-linked sequences will be uncommon. For the purpose of reconstructing species phylogenies, this would be an advantageous property, because attempts to reconstruct historical relationships among species using Y-linked loci would not be confounded by reticulation. A4us Phylogenetic Relationships The total evidence tree combining data from Sry, mtDNA, and allozymes (fig. 2 D) has more explanatory power than trees based on any data set considered alone. The phylogeny is concordant with Marshall’s ( 1977, 198 1) classification, which is based primarily on morphological data and karyotypes, and with the single-copynuclear DNA hybridization tree presented in She et al.

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RICHARD HARRISON, reviewing Received

August

16, 1993

Accepted January 9, 1994

editor