Biochemical Genetics, Vo128,Nos. 3/4, 1990

Differentiation of Restriction Sites in Ribosomal DNA
in the Genus Apodemus
Hitoshi Suzuki, 1'2 Kimiyuki Tsuchiya, 3 Mitsuru Sakaizumi, 4 Shigeharu
Wakana, 50samu Gotoh, 6 Naruya Saitou, 7 Kazuo Moriwaki, 8 andSusumu Sakurai 1

Received 22 June 1989--Final 11 Dec. 1989

Southern blot analysis of ribosomal DNA (rDNA) from seven species of
A p o d e r n u s was carried out in order to examine the genetic relationships
between the species. Analysis of heterogeneity in rDNA spacers in A. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, and A.
peninsulae, using 13 different restriction enzymes and cloned mouse rDNA
probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction
sites, sequence divergence among the different major repetypes was estimated. Among the seven species o f A p o d e m u s examined, the major repetypesof A. flavicollis and A. sylvaticus were the most closely related, having only
1.0% sequence divergence. These repetypes and those of the remaining fivespecies differ substantially from one another, with 4.3-8.5 % divergence.

This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of
Education, Science and Culture, Japan.
Division of Molecular Genetics, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi,
Minato-ku, Tokyo 105, Japan.
2 To whom correspondence should be addressed.
3 Experimental Animal Center; Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan.
4 Department of Experimental Animal Science, Tokyo Metropolitan Institute of Medical Science,
3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan.
5 Department of Genetics, Central Institute for Experimental Animals, Nogawa, Miyamae-ku,
Kawasaki 213, Japan.
6 Department of Biochemistry, Saitama Cancer Center Research Institute, Kitaadachi-Gun,
Saitama, 362, Japan.
7 Department of Anthropology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113, Japan.
8National Institute of Genetics, Mishima 411, Japan.
137
0006-2928/90/0400-0137506.00/0© 1990 Plexaum Publishing Corporation

138

Suzuki et ai.

KEY WORDS:wood mice; Apodemus; ribosomal DNA (rDNA); restriction fragment length
polymorphism(RFLP);sequencedivergence.
INTRODUCTION
Wood mice (Apodemus, Muridae), of which there are 13 known species, arethe most common small rodents living in fields and mountains around the Old
North region (Corbet, 1978). Unlike house mice or rats, whose migration
depends largely on human actions, dispersal of wood mice is thought to berather independent of such influences. Thus, analysis of genetic differences
between isolated populations may provide useful data for evolutionary biology
and zoogeography. Although Apodemus species have been compared in termsof morphology, karyology, and biochemical genetics (Imaizumi, 1962, 1964;
Kral, 1970; Tsuchiya, 1974; Bekasova et al., 1980; Bonhomme et al., 1984),
there is no consensus as to their phylogenetic relationships. In the last decade,qualitative genetic analysis of related species has been performed at the DNA
level based on restriction enzyme fragment length polymorphism (RFLP), forcytoplasmic mitochondrial DNA (mtDNA) (Yonekawa et al., 1981; Ferris etal., 1983) and nuclear genomic rDNA (Arnheim et al., 1980; Wilson et al.,
1984; Hillis and Davis, 1986, 1988; Hillis, 1987; Suzuki et al., 1986, 1987).
The rRNA locus exists as a multigene family which consists of several
hundred copies in the mammalian genome. The copies are usually separated
into tandemly repeated clusters at several different sites on chromosomes.
Each rDNA repeating unit is composed of three rRNA genes, namely, thosefor 28, 5.8, and 18 S RNA, which are separated from each other by spacers.
The spacers are known to evolve rapidly (Arnheim, 1983), and particularly
rapid evolution is observed in nontranscribed spacers (NTS), where RFLPs
are generated predominantly by base changes at restriction sites and thedeletion, insertion, or duplication of DNA segments.
We constructed restriction maps of an approximately 40-kb portion of
the repeating units of Apodemus rDNA, encompassing the rRNA coding
region, the internal spacer regions, and the flanking spacer regions, using four
different probes. In this report, we compare the major repetypes of sevenspecies of Apodemus and discuss the genetic relationships among the sevenspecies.
MATERIALS AND METHODS
Wood Mice

A total of 18 individual mice from seven species of Apodemus was used (Table
I). Wood mice were trapped and kept at the Experimental Animal Center ofMiyazaki Medical College prior to experiments.

139

Restriction Sites in rDNA in Apodemus
Table I. List of Samples Used

ApodemusspeciesA. sylvaticus
A. flavicollis
A. semotus
A. agrarius
A. argenteus
A. speciosus
A. peninsulae

Locality

No. of mice
examined

1. Bonn(West Germany)
2. Liibeck(West Germany)
3. Mt. Lisan (Taiwan)
4. Liibeck(West Germany)
5. Jindo Island (Korea)
6. Miyazaki (Japan)
7. Fukushima (Japan)
8. Piacol (Korea)
9. Hokkaido(Japan)

2
1
4
1
4
1
3
1
1

Blot Analysis

Nuclear DNAs were prepared from the livers of wood mice as described byManiatis et al. (1982). The DNAs were digested with restriction enzymes andsubjected to electrophoresis on 0.6-0.7% agarose gels at 2 V / c m for 13-15 hr
in 40 mM Tris-acctate buffer ( p H 7.8) that contained 2 mM EDTA. Doublestranded fragments of D N A were transferred to nylon filters, baked at 80°C
for 2 hr, and hybridized with a probe as described by Southern (1975).
Labeling of probes was carried out using (a-32p)-dCTP and random primers.
The specific radioactivity of probes was 4-8 × 108 cpm/lzg. Prior to hybridization, the filters were incubated at 66°C for 2-4 hr in 6× SSC (1 × SSC = 0.15
M NaC1-0.015 M Na citrate at p H 7.0) which contained 0.01% (w/v) sodium
dodecyl sulfate and Denhardt's solution with denatured salmon sperm D N A(100 #g/ml). Hybridization was performed at 66°C for 20 hr, and filters were
washed twice with 0.1 × SSC for 30 min at room temperature and thenwrapped in Saran wrap (Dow Chemicals) to prevent drying. Autoradiography
was performed on Fuji-RX film for 12-24 hr at room temperature with anintensifying screen. Then filters were boiled in distilled water at 95°C for 5-10
rain to remove the probe DNA. After air-drying the filters were reused forhybridization with another probe.
Construction of Restriction Maps

To construct the restriction maps for the various types of rDNA repeating unit
(repetypes), the genomic DNAs from seven species of Apodemus, A. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, and A.
peninsulae, were subjected to single and double digestion with 13 restriction
enzymes, namely, AatI, ApaI, BamHI, BglII, DraI, EcoRI, EcoRV, HindIII,
KpnI, PstI, PvuII, SacI, and XbaI. The digested DNAs on nylon filters were140

Suzukiet al.

hybridized sequentially with four 32p-labeled rDNA probes, "18SA," "18SB,""28S," and "INT." These probes were prepared from clones of mouse rDNA
according to Kominami et a l. (1981, 1982). The specific probes are indicatedin Fig. 2.
Since the sites of cleavage by EcoRI sites on the genes for 18 and 28 S
rRNA, designated E1 and E2 in Fig. 2, were judged to be conserved in
Apodemus rDNA, as they are in most mammalian species, hybridization data
after double digestion with EcoRI plus another enzyme were usually quite
useful for the construction of restriction maps.
Construction of Phylogenetic Trees
To estimate the sequence divergence among seven major repetypes represented in Fig. 2, we compared the arrangement of restriction sites between
pairs of rDNA repetypes and counted common and different sites. Employing
a method developed by Gotoh et al. (1979) in which backward mutations andparallel mutations are taken into account (Jukes and Cantor, 1969), we
produced a matrix of sequence divergences among all possible combinations ofrepetypes (Table II). Then we constructed phylogenetic trees using theunweighted pair-group (UPG) method (Sokal and Sneath, 1963) and theneighbor-joining (N J) method (Saitou and Nei, 1987). Since insertions anddeletions of small DNA fragments frequently occur in rDNA spacers, weselected informative restriction sites near the two probes (see Fig. 2) andconducted a phylogenetic analysis using the maximum parsimony (MP)method (Fitch, 1971) employing a computer program developed by Saitou
and Imanishi (1989).
RESULTS
Restriction Maps of Major rDNA Repetypes from Seven Species of
apodemusFrom the patterns of hybridization after single and double digestions, we first
constructed restriction maps for the coding and internal spacer regions ofgenes for rRNA. Most restriction sites in the coding region were conserved,
indicating little significant differentiation within each species and also among
the seven species examined. Exceptionally, digestion with SacI and ApaI of
the gene for 18 S rRNA showed some difference between species, but thegenes were almost monomorphic within each genome and within each species.
Since E l / E 2 fragments from the seven species were digested by ApaI into
several small segments, we could not localize the ApaI sites within E1/E2. For
all samples investigated, DraI and KpnI sites in the internal spacers were also

Restriction Sites in rDNA in Apodemus

141

Table II. Sequence Divergence Among the Major rDNA Repetypes of Seven Species of
Apodemus (Upper Right), Based on the Number of Common and Different Restriction Sites(Lower Left)
Sequence divergence (%)
Species

syl.

fla.

sere.

agra.

arg.

spe.

pen.

1. sy&aticus

--

1.0

4.7

6.1

7.2

8.5

8.5

11/1
4/0
10/2
25/3

--

5.1

6.8

7.2

8.5

7.2

8/4
3/1
6/6
17/11

7/6
3/1
7/5
17/12

4.3

6.8

8.1

7.2

7/5
3/2
6/7
16/14

6/6
3/2
6/7
15/15

8/5
3/1
8/5
19/11

8.1

6.8

6.8

6/6
2/4
7/6
15/16

6/6
2/4
7/6
15/16

6/6

4/9

6.8

6.8

7/6
15/15

6/6
13/16

5/8
2/3
6/6
13/17

4/9
2/3
7/5
13/17

4/9

5/8

7/5
13/16

8/5
15/15

5/8
2/2
6/7
13/17

5/8
2/2
7/5
14/15

6/7
2/1
6/7
14/15

5/7
2/1
7/6
14/14

2. flavicoll&

a~
b
c
Total
3. semotus

a
b
c
Total
4. agra~us

a
b
c
Total
5. argenteus

a
b
c
Total

2/3

3/1

6. speciosus

a
b
c
Total

2/2

2/2

5/8

8.1

3/1
7/6
15/15

7. peninsulae

a
b
c
Total

7/6

4/9

6/7
15/15

7/6
13/16

2/2

2/1

~Number of sites (common/different) for the spacer upstream from the gene for 18 S rRNA (a),the internal spacer region (b), and the NTS region downstream from the gene for 28 S rRNA (c).
observed in the corresponding regions of samples from house mice and rats
(data not shown).
By reference to the maps of the coding and the internal spacer regions,the location of the restriction sites on the external spacer regions, whichflanked the genes for 18 and 28 S r R N A , was estimated by hybridization,
after single digestion, with the 18 SB and 28 S probes, both of which are

142

Suzuki et al.

localized to the distal end of the coding regions. The arrangement of restriction sites was confirmed by hybridization after double digestions (see Fig. 1).
The restriction maps of the major rDNA repetypes are presented schematically in Fig. 2. These maps display only the nearest restriction sites on theexternal spacers which flank the genes for 18 or 28 S RNA.
Although length polymorphisms within the genome were observed incertain regions of the external spacer in most samples investigated, only themost prominent bands were taken into account for construction of the physicalmaps. Some restriction sites also showed heterogeneity within a genome,
within a population, and between populations of the same species. Then we
arranged the restriction sites that provided evidence for the most predominant
repetypes in each species. Significant variations with respect to the presence orabsence of certain restriction sites were observed in the case of some restriction sites among individuals from the same locality. Such sites are marked on
the restriction maps in Fig. 2 with asterisks.
In the present study, we analyzed two populations [Jindo (Korea) and
Liibeck (West Germany)] of A. agrarius. We found that a variation in
restriction sites on rDNA was present in the AatI site in the internal spacer.
The AatI site was observed in 72% of rDNA units for Jindo samples on
average and in 22% in the case of the Ltibeck sample. In the case of A.
peninsulae, no significant difference in the arrangement of major restriction
sites was observed between the two individuals collected from Piacol (Korea)
and Hokkaido (Japan), which have been taxonomically classified as belonging
to different subspecies of A. peninsulae, namely, A. p. peninsulae and A. p.
giliacus (Kobayashi and Hayata, 1971).

DISCUSSION
In the present study, we investigated the phylogenetic relationships among
seven species of Apodemus based on the differentiation of rDNA sequences. It
was clear that multiple mutations, including mutations in size and at restriction sites, had accumulated in rDNA, especially in its spacer regions. However, most of the mutations appeared to be have been fixed to yield specificrepetypes within populations or species during the course of differentiation.
Changes in such a manner, designated as evidence of concerted evolution,
have been well documented in families of repetitive DNA, especially in the
rDNA family (see Dover, 1982, for review). In this study we were easily able
to characterize the rDNA family of each species with single restriction mapsof the major repetypes and we used these maps as an index of genetic diversity
among the seven species examined. To estimate the degree of the sequence
divergence between repetypes, comparisons of site differences are very useful(Gotoh et al., 1979). Accordingly, we neglected differences in size, and only

Restriction Sites in rDNA in Apodemus

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Restriction Sites in rDNA inApodemus

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differences in the arrangement of restriction sites along the restriction maps ofthe rDNA spacers were taken into account for estimations of the degree ofdivergence among the repetypes. It should be noted that the values used toquantitate the divergence of sequences (Table II) are somewhat speculative
because site rearrangements are generated not only by point mutations but
also by segment mutations which include deletion, insertion, duplication andinversion.
From the estimates of the amount of sequence divergence, we constructed
two hypothetical phylogenetic trees for these repetypes, by the UPG and NJ
methods (Figs. 3a and b). We also constructed a phylogentic tree by the MP
method. Restriction sites were used as characters and informative sites wereselected from the restriction maps as listed in Table III. We obtained two
equally parsimonious tree topologies which were similar to those by the UPG
and NJ methods (Fig. 3). Based on these analysis, we now propose a new set ofcriteria for the classification of our seven species of Apodemus. The averagesequence divergence of six repetypes (Nos. 2-7 in Table II) was 7.0%, whilethat between the human and the Japanese macaque (Macaca fuscata) andbetween Mus musuculus domesticus and M. m. molossinus are 7.3 and 1.5%,
respectively, when analyzed under the same conditions as those used here for
Apodemus (Suzuki et al., unpublished data). The divergence of human from
macaque and of M. m. domesticus from M. m. molossinus is considered tohave occurred 2-3 x 107 (Hasegawa et al., 1988) and 1-2 x 106 years ago(Yonekawa et al., 1981), respectively. Extrapolating from these data, we canassume that divergence of the various species of Apodemus occurred approximately 107 years ago. This value agrees with Bonhomme's (1984) suggestionthat A. agrarius is distantly related to A. sylvaticus (1984).
Ribosomal DNA spacer difference indicates that A. sylvaticus and A.
flavicollis are the most closely related species among the seven species
investigated. A sequence divergence between the major rDNA repetypes of
two species of 1% is similar to that between the two mouse subspecies
described above and to that between Rattus rattus and R. norvegicus (Suzuki
et al., unpublished data). The close relationship between A. sylvaticus and A.
flavicollis is also supported by analyses of karyotypes (Kral, 1970) isozymes(Gebczynski et al., 1986), satellite DNA (Brown and Dover, 1979) and
mtDNA (Wakana et al., manuscript in preparation). Although Gebczynski etal. (1986) estimated the divergence of A.flavicollis from A. sylvatieus to haveoccurred 15-30 x 10 4 years ago, based on their biochemical analysis, ourresults indicate that the divergence occurred 67-133 x 10 4 years ago. In
conclusion, our results suggest the following process of species differentiation.
The ancestors of the seven species first differentiated into five groups, sylvaticus/
flavicollis, semotus/agrarius, argenteus, speciosus, and peninsulae. Then,semotus and agrarius were separated. About 1 million years ago sylvaticus

146

Suzuki et al.

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Restriction Sites in rDNA in Apodemus

147

Table III. Presence (+) or Absence ( - ) of Hypothetical Common Restriction Sites Within the
Major rDNA Repetypes of Seven Species of Apodemus
Restriction sitea
Species

1. syl.

2. fla.
3. sere.

4. agr.
5. arg.
6. spe.
7. pen.
Informativesite

D V P X G A K S Z Z S A S X Z KAZ E D S X B H V P+++--+++++--+++ + - + + + - - - ++ + + - - + + + + ++----+++--+++------++--+--+++--++--+------+----+++++ +-- ++----+++--+++--++-+-----++ ++ ++--++++++---++--++----+++--+--++----++ +------+++ +--+++--++++++--+++++--+--+
+
00000

00

00000

00000

0000

aRestriction sites shared by more than one repetype between DraI sites upstream of the 18 S
rRNA gene and the Pstl sites downstream of the 28 S rRNA gene were analyzed (see Fig. 2).
The hypothetical common sites were selected to maximize the number of pluses in this table.
Abbreviations of the restriction sites and their arrangement are as in the legend to Fig. 2.

andflavicollis were differentiated from their common ancestor. Our results do
not provide any genetic basis for the classification into two "ecotypes," i.e., adivision into field types (agrarius, flavicollis, peninsulae, and speciosus) andwood types ( sylvaticus, sernotus, and argenteus ).
ACKNOWLEDGMENTS
We thank Drs. R. Kominami and M. Muramatsu for providing us with mouse
rDNA clones. We also thank Drs. H. Winking and Y. Harada for their kind
help in collection of wild wood mice. We wish to thank Miss R. Nakayama forher valuable technical assistance.

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