The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome

Abstract

The genus Tokudaia comprises three species, two of which have lost their Y chromosome and have an XO/XO sex chromosome constitution. Although Tokudaia muenninki (Okinawa spiny rat) retains the Y chromosome, both sex chromosomes are unusually large. We conducted a molecular cytogenetic analysis to characterize the sex chromosomes of T. muenninki. Using cross-species fluorescence in situ hybridization (Zoo-FISH), we found that both short arms of the T. muenninki sex chromosomes were painted by probes from mouse chromosomes 11 and 16. Comparative genomic hybridization analysis was unable to detect sex-specific regions in the sex chromosomes because both sex probes highlighted the large heterochromatic blocks on the Y chromosome as well as five autosomal pairs. We then performed comparative FISH mapping using 29 mouse complementary DNA (cDNA) clones of the 22 X-linked genes and the seven genes linked to mouse chromosome 11 (whose homologue had fused to the sex chromosomes), and FISH mapping using two T. muenninki cDNA clones of the Y-linked genes. This analysis revealed that the ancestral gene order on the long arm of the X chromosome and the centromeric region of the short arm of the Y chromosome were conserved. Whereas six of the mouse chromosome 11 genes were also mapped to Xp and Yp, in addition, one gene, CBX2, was also mapped to Xp, Yp, and chromosome 14 in T. muenninki. CBX2 is the candidate gene for the novel sex determination system in the two other species of Tokudaia, which lack a Y chromosome and SRY gene. Overall, these results indicated that the Y chromosome of T. muenninki avoided a loss event, which occurred in an ancestral lineage of T. osimensis and T. tokunoshimensis, through fusion with an autosome. Despite retaining the Y chromosome, sex determination in T. muenninki might not follow the usual mammalian pattern and deserves further investigation.

Full text

The Y chromosome of the Okinawa spiny rat, Tokudaia
muenninki, was rescued through fusion with an autosome
Chie Murata &Fumio Yamada &
Norihiro Kawauchi &Yoichi Matsuda &
Asato Kuroiwa
Published online: 24 December 2011
#Springer Science+Business Media B.V. 2011
Abstract The genus Tokudaia comprises three species,
two of which have lost their Y chromosome and have an
XO/XO sex chromosome constitution. Although Toku-
daia muenninki (Okinawa spiny rat) retains the Y chro-
mosome, both sex chromosomes are unusually large. We
conducted a molecular cytogenetic analysis to character-
ize the sex chromosomes of T. mu e n n i n k i. Using cross-
species fluorescence in situ hybridization (Zoo-FISH),
we found that both short arms of the T. muenninki sex
chromosomes were painted by probes from mouse chro-
mosomes 11 and 16. Comparative genomic hybridization
analysis was unable to detect sex-specific regions in the
sex chromosomes because both sex probes highlighted
the large heterochromatic blocks on the Y chromosome
as well as five autosomal pairs. We then performed
comparative FISH mapping using 29 mouse complemen-
tary DNA (cDNA) clones of the 22 X-linked genes and
the seven genes linked to mouse chromosome 11 (whose
homologue had fused to the sex chromosomes), and
FISH mapping using two T. muenninki cDNA clones of
the Y-linked genes. This analysis revealed that the ances-
tral gene order on the long arm of the X chromosome and
the centromeric region of the short arm of the Y chromo-
some were conserved. Whereas six of the mouse chro-
mosome 11 genes were also mapped to Xp and Yp, in
addition, one gene, CBX2, was also mapped to Xp, Yp,
and chromosome 14 in T. muenninki.CBX2 is the candi-
date gene for the novel sex determination system in the
two other species of To k u d a i a , which lack a Y chromo-
some and SRY gene. Overall, these results indicated that
the Y chromosome of T. muenninki avoided a loss event,
whichoccurredinanancestrallineageofT. o s i m e nsis and
T. tokunoshimensis, through fusion with an autosome.
Despite retaining the Y chromosome, sex determination
Chromosome Res (2012) 20:111–125
DOI 10.1007/s10577-011-9268-6
Responsible Editor: Tariq Ezaz and Jennifer Graves.
C. Murata :A. Kuroiwa
Graduate School of Life Science, Hokkaido University,
Kita 10 Nishi 8, Kita-ku,
Sapporo, Hokkaido 060-0810, Japan
F. Yamada
Forestry and Forest Products Research Institute,
Tsukuba, Ibaraki 305-8687, Japan
N. Kawauchi
Yachiyo Engineering Co., Ltd,
Kokuba Bldg. 9f, 3-21-1, Kumoji,
Naha, Okinawa 900-0015, Japan
Y. Matsuda
Laboratory of Animal Genetics, Department of Applied
Molecular Biosciences, Graduate School of Bioagricultural
Sciences, Nagoya University,
Furo-cho, Chikusa-ku(Nagoya 464-8601, Japan
A. Kuroiwa (*)
Laboratory of Animal Cytogenetics, Faculty of Science,
Hokkaido University,
Kita 10 Nishi 8, Kita-ku,
Sapporo, Hokkaido 060-0810, Japan
e-mail: asatok@cris.hokudai.ac.jp

in T. mu e n n i nki might not follow the usual mammalian
pattern and deserves further investigation.
Keywords X chromosome .evolution .Zoo-FISH .
FISH .CBX2 .Spiny rat .Tokudaia
Abbreviations
ASA American Standards Association
BSA Bovine serum albumin
cDNA Complementary DNA
CGH Comparative genomic hybridization
dUTP Deoxyuridine 5-triphosphate
FISH Fluorescence in situ hybridization
FITC Fluorescein isothiocyanate
HMG High-mobility group
IHB Intercalary heterochromatin block
kb Kilo base pairs
MMU Mus musculus chromosome
MYA Million years ago
ORF Open reading frame
PAR Pseudoautosomal region
qRT-PCR Quantitative real-time PCR
RBMY1A1 RNA binding motif protein, Y
chromosome, family 1, member A1
SSC Saline sodium citrate
TMU Tokudaia muenninki chromosome
TTO Tokudaia tokunoshimensis chromosome
UV Ultraviolet
Zoo-FISH Cross-species fluorescence in situ
hybridization
Introduction
The genus Tokudaia belongs to the subfamily
Murinae (Muridae, Rodentia) and consists of three
species: Tokudaia muenninki (Okinawa spiny rat),
Tokudaia osimensis (Amami spiny rat), and Toku-
daia tokunoshimensis (Tokunoshima spiny rat).
Each species is endemic to an island in the south-
ernmost part of Japan and is named after the
island on which it is found. The sex chromosomes
in this genus are unusual among mammals. The diploid
chromosome numbers of T. osimensis and T. to k u n o shi-
mensis are an odd number, 25 and 45, respectively.
Neither T. osimensis nor T. tokunoshimensis has a Y
chromosome and sex is determined with an XO/XO
sex chromosome constitution in both species (Honda
et al. 1977,1978; Kobayashi et al. 2007). Although the
diploid chromosome number of T. muenninki is 44 with
XX/XY sex chromosome constitution, both sex chro-
mosomes are unusually large (Tsuchiya et al. 1989;
Murata et al. 2010). The euchromatic regions of the X
and Y chromosomes occupy 8% and 4% of the haploid
genome, respectively, which suggests that an autosomal
addition has occurred (Murata et al. 2010). On the basis
of molecular phylogenetic analysis, T. m u e n ninki was
the first species in the genus to diverge, which suggests
that the Y chromosome was lost in the common ancestor
of T. osimensis and T. tokunoshimensis after this event
(Murata et al. 2010). Three stepwise events that led to the
disappearance of the Y chromosome have been sug-
gested; gene transpositions from the Y chromosome to
autosomes, superseding Y-linked genes by new genes on
the X chromosome or autosomes, and Y-to-X transloca-
tion containing Y-linked genes (Arakawa et al. 2002;
Kuroiwa et al. 2010).
The SRY gene, which is the master gene for male
sex determination in placental mammals (Sinclair et al.
1990; Koopman et al. 1991) and is located on the Y
chromosome, has been lost completely in T. osimensis
and T. tokunoshimensis (Soullier et al. 1998; Sutou et
al. 2001). Other Y chromosome genes have also been
lost in T. osimensis, including RBMY1A1 (RNA bind-
ing motif protein, Y chromosome, family 1, member
A1) (Kuroiwa et al. 2010), which plays a role in
spermatogenesis (Mazeyrat et al. 1999; Elliott 2004).
However, most proto-Y-linked genes have been con-
served in both sexes of T. osimensis and T. tokunoshi-
mensis through translocation from the Y chromosome
to the distal region of the long arm of the X chromo-
some. The absence of SRY indicates that T. osimensis
and T. tokunoshimensis must have a novel sex-
determining mechanism. Recently, CBX2 (chromobox
homolog 2, synonym is M33) has emerged as a can-
didate gene for sex determination in these species
(Kuroiwa et al. 2011). CBX2 is a single copy gene
that is located on an autosome in mice and humans.
However, quantification of the copy number of CBX2
in T. osimensis and T. tokunoshimensis by quantitative
real-time PCR showed that there were two or three
more copies of CBX2 per haploid genome in males
than in females, which suggests that CBX2 might be
involved in a novel sex-determining mechanism
through gene dosage (Kuroiwa et al. 2011).
Although T. muenninki possesses a Y chromosome,
several aspects of sex determination in this species are
112 C. Murata et al.

unusual (Tsuchiya et al. 1989;Murataetal.2010).
Multiple copies of the SRY gene (>70) are found
in T. muenninki, and these are distributed along the
entire long arm of the Y chromosome (Murata et
al. 2010). Most copies are not functional; however,
an open reading frame (ORF) is conserved in three
copies (Murata et al. 2010). A single amino acid
substitution that is specific to T. muenninki was
detected in the DNA binding surface domain in
the high-mobility group (HMG)-box of all SRY
copies (Murata et al. 2010). This region is impor-
tant for the binding of SRY to target DNA sequences,
which suggests that the DNA-binding ability of all SRY
proteins produced in T. muenninki is weakened by this
mutation.
To reveal the composition of the X and Y chromo-
somes in T. muenninki, we performed cross-species
fluorescence in situ hybridization (Zoo-FISH) with
chromosome-specific DNA probes constructed from
the laboratory mouse (Mus musculus). To describe the
composition in more detail and identify sex-specific
regions in the sex chromosomes, we performed com-
parative genomic hybridization (CGH) analysis.
When this provided limited information, we then
conducted comparative FISH analysis using 29
mouse cDNA clones of functional genes linked to
the X chromosome and chromosome 11 and FISH
mapping using two T. muenninki cDNA clones of
the Y-linked genes.
Materials and methods
Sample collection, cell culture, and DNA and RNA
extraction
The three species of Tokudaia are all endangered (The
IUCN Red List of Threatened Species; http://www.
iucnredlist.org/ 15/9/2011) and have been protected
by the Japanese government as natural treasures since
1972. With permission from the Agency for Cultural
Affairs and the Ministry of the Environment in
Japan, we trapped the Okinawa spiny rat, T. mu e n -
ninki, on Okinawa-jima Island in March 2008 and
February 2009 (Yamada et al. 2010). We collected
tail tissue from four males and three females for use
in the study. Cell culture and DNA extraction were
performed as described by Murata et al. (2010).
Total RNA was extracted from fibroblasts derived
from tail tissue from a T. mu e n n inki male using
TRIZOL Reagent (Invitrogen) in accordance with
the manufacturer’s protocol. The cDNAs were syn-
thesized using SuperScript II Reverse Transcriptase
(Invitrogen).
DNA probes
Seven mouse cDNA clones of functional genes
linked to chromosome 11 and 22 linked to the X
chromosome were used in the study. Their gene
names, symbols, accession numbers, sizes, and
chromosomal location in mouse (M. musculus), rat
(Rattus norvegicus), and three Tokudaia species are
listed in Table 1.A 6.6-kilo base pairs (kb) mouse
genomic DNA fragment was used for chromosomal
localization of the 18S–28S rRNA gene (Kominami
et al. 1982).
Clones of T. muenninki DDX3Y (AB672502;
1.8 kb) and UTY (AB672501; 2.0 kb), which are both
linked to the Y chromosome, were also used for FISH
mapping. The mouse homologues referred to here are
Ddx3y (NM_012008) and Uty (NM_009484), and the
rat genes are DDX3Y (FJ775727) and UTY
(FJ775728). The T. muenninki homologues of these
genes were cloned by degenerate PCR. The primer
pairs used in the PCR were as follows: DDX3Y,5′-
TGA TCG TGG AAG TGG ATC C-3′and 5′-CAC
AGC TGG TAA TGT ACT GCA-3′; UTY, 5′-GAC
GCT GTT GAA CAA GGC AA-3′and 5′-GGA GTG
GGT GGA TTC AAC TTC-3′. The PCR for DDX3Y
was carried out in 10 μlof1×ExTa q buffer that
contained 0.5 μl of template cDNA, 2 mM MgCl
2
,
0.5 μM of each primer, 0.2 mM each of the four
dNTPs,and0.25UofExTaq (TaKaRa Bio Inc.).
The reaction profile was denaturation for 5 min at
94°C, followed by 35 cycles of denaturation for 30 s
at 94°C, annealing for 30 s at 60 °C, and extension for
2 min at 72°C, and then a final extension at 72°C for
7 min. The PCR for UTY was carried out in 20 μlof
1× P r i meSTAR G X L b uffer ( M g
2+
plus) that
contained 1.0 μl of template cDNA, 0.3 μM of each
primer, 0.2 mM each of the four dNTPs, and 0.25 U of
PrimeSTAR GXL DNA Polymerase (TaKaRa Bio
Inc.). The reaction profile was denaturation for 3 min
at 94°C, followed by 35 cycles of denaturation for 10 s
at 98°C, annealing for 15 sec at 60°C, and extension
for 1.5 min at 68°C, and then a final extension at 72°C
for 5 min.
Sex-autosome fusions in Tokudaia muenninki 113

Table 1 The list of mouse cDNA clones used for FISH mapping and their chromosomal locations
Gene
symbol
Gene name cDNA clone Chromosome location
Size (kb) Mouse
a
Rat
a
T. muenninki
d
T. osimensis
e
T. tokunoshimensis
e
STK10 Serin/threonine kinase 10 3.1 11 A4
b
10q 12.3 Xp, Yp ––
ADAM19 A disintegrin and
metalloproteinase
domain 19 (meltrin beta)
1.4 11 A5-B1.1
c
10q 21.3-q 22 Xp, Yp ––
DOC2 βDouble C2 beta –11 B3-B5 10q Xp, Yp ––
COASY Coenzyme A synthase 2.0 11 D 10q 32.1 Xp, Yp ––
GAA Glucosidase, alpha, acid 2.0 11 D-E 10 Xp, Yp ––
CBX2 Chromobox homolog 2
(Drosophila Pc class)
1.5 11 E2 10q 32.3 Xp, Yp, 14 3q 24, 6p 11.2
f
10q 25-26,
14q 12-13.1
f
ARHGDIA Rho GDP dissociation
inhibitor (GDI) alpha
–11 E2 10q 32.3 Xp, Yp ––
ARAF v-raf murine sarcoma 3611
viral oncogene homolog
1.2 X A2-A3.1 Xq 11.1 Xq Xp 11.3 Xq 11
ELK1 ELK1, member of ETS
oncogene family
1.2 X A2-A3.1 Xq 11.1 Xq Xp 11.3 Xq 11
PDHA1 Pyruvate dehydrogenase
E1 alpha 1
0.9 X A4 Xq 11.1 Xq Xp 11.3 Xq 11
TSPAN7 tetraspanin 7 2.0 X A1.3-A2 Xq 11.2-12 Xq Xp 11.2-11.3 Xq 11-12
MAOA Monoamine oxidase A 1.4 X A2 Xq 12 Xq Xp 11.12-11.2 Xq 12
PLP2 Proteolipid protein 2 1.2 X A2-A3.1 Xq 12 Xq Xp 11.12-11.2 Xq 12
GATA1 GATA binding protein 1 1.0 X A2 Xq 12 Xq Xp 11.12-11.2 Xq 12
PIGA Phosphatidylinositol glycan
anchor biosynthesis, class A
1.1 X F5 Xq 12-13 Xq Xp 11.12 Xq 12
SAT1 Spermidine/spermine
N1-acetyl transferase 1
1.1 X F3-F4 Xq 13-14 Xq Xp 11.11-11.12 Xq 12-13.1
PHKA2 Phosphorylase kinase alpha 2 2.3 X F3-F4 Xq 13-14 Xq Xp 11.11-11.12 Xq 12-13.1
DXHXS423 DNA segment, Chr X,
human DXS423
0.8 X C3 Xq 21.3 Xq Xq 11 Xq 13.3
EFNB1 Ephrin B1 1.0 X D Xq 22.1 Xq Xq 12 Xq 21
PGK1 Phosphoglycerate kinase 1 1.5 X D Xq 22.3-31.1 Xq Xq 12-21 Xq 21-22
PLP1 Proteolipid protein (myelin) 1 1.5 X F1-F2 Xq 32 Xq Xq 21-22 Xq 22-23
HTR2C 5-Hydroxytryptamine
(serotonin) receptor 2 C
1.4 X F1 Xq 34-35.1 Xq Xq 23.1 Xq 24.1
SLC25A5 Solute carrier family 25
(mitochondrial carrier,
adenine nucleotide
translocator), member 5
1.4 X A4 Xq 36 Xq Xq 23.1 Xq 24.1
FHL1 Four and half LIM
domains 1
2.0 X A6-A7.1 Xq 36 Xq Xq 23.2 Xq 24.2
IDH3G Isocitrate dehydrogenase
3 (NAD+) gamma
1.3 X A7.3-B Xq 37.1-37.2 Xq Xq 23.3 Xq 24.3
PLXNA3 Plexin A3 1.3 X B-C1 Xq 37.1-37.2 Xq Xq 23.3 Xq 24.3
CETN2 Centrin 2 0.8 X B Xq 37.1-37.2 Xq Xq 23.3 Xq 24.3
GDI1 Guanosine diphosphate
(GDP) dissociation
inhibitor 1
1.1 X B-C1 Xq 37.1-37.2 Xq Xq 23.3 Xq 24.3
BGN biglycan 1.7 X B Xq 37.2 Xq Xq 23.3 Xq 24.3
a
Kuroiwa et al. (1998)
b
Kuramochi et al. (1999)
c
Kurohara et al. (2000)
d
Present study
e
Kobayashi et al. (2008)
f
Kuroiwa et al. (2011)
114 C. Murata et al.

Chromosome preparation and Zoo-FISH analysis
The preparation of R-banded chromosomes was per-
formed as described by Matsuda et al. (1992a), Matsuda
and Chapman (1995), and Kobayashi et al. (2008).
The Zoo-FISH analysis was performed using
biotin- and Cy3-labeled chromosome-specific painting
probes from the laboratory mouse (Cambio Ltd.).
FISH was carried out as described by Nakamura et
al. (2007) with slight modifications. The probes were
denatured at 75°C for 10 min and pre-annealed by
incubation at 37°C for 1 h. The chromosome slides
were hybridized with the biotin- or Cy3-labeled
probes at 37°C for 5 days. After hybridization with
the biotin-labeled probes, the slides were washed in
50% formamide in 2× saline sodium citrate (SSC) at
37°C for 15 min, in 2× SSC for 15 min, and then in
4× SSC for 5 min at room temperature. To detect the
hybridization signals of the biotin-labeled probes, the
chromosome slides were incubated with avidin fluo-
rescein isothiocyanate (FITC) (Vector Laboratories) or
streptavidin-Cy5 (GE Healthcare UK Ltd.) diluted
1:500 in 1% BSA/4× SSC at 37°C for 1 h. The slides
were washed sequentially on a shaker with 4× SSC,
0.1% Nonidet P-40/4× SSC, and then 4× SSC for
5 min each at room temperature. After hybridization
with the Cy3-labeled probe, the slides were washed
sequentially in 4× SSC, 0.1% Nonidet P-40/4× SSC,
and then 4× SSC for 5 min each at room temperature.
CGH analysis
The CGH analysis was conducted using metaphase
spreads from one male and one female of T. mu e n -
ninki as described by Kobayashi et al. (2007)with
slight modifications. The genomic DNA for the
probes was also extracted from one male and one
female. The male and female genomic DNA probes
were labeled differently, with Cy3-deoxyuridine
5-triphosphate (dUTP; GE Healthcare) and fluorescein-
12-dUTP (Invitrogen), respectively, by nick translation
at 15°C for 90 min. The labeled DNA probes were
denatured at 75°C in formamide and dissolved in
hybridization buffer (50% formamide, 2× SSC, 10%
dextran sulfate, and 2 mg/ml BSA). The cocktail of
probes for one slide contained 700 ng of labeled DNA
from the female and 700 ng of labeled DNA from the
male. Slides were hybridized at 37°C for 4 days. The
slides were then washed sequentially in 4× SSC, 0.1%
NonidetP-40in4×SSC,4×SSC,and2×SSCfor5min
each at room temperature, and then rinsed in 2× SSC.
FISH analysis
The FISH analysis using cDNA clones as probes was
performed as described by Kuroiwa et al. (2010). The
exception was the FISH analysis using the 18S–28S
rRNA gene clone as a probe. In this case, the chromo-
some slides were treated with RNase (1 μg/μlin2×
SSC) for 1 h, washed three times in 2× SSC for 3 min
each, and dehydrated in 70% and 100% ethanol at 4°C
for 5 min each before the chromosomes were dena-
tured in 70% formamide in 2×SSC at 69°C for 2 min.
After hybridization and washing, the slides were incu-
bated under parafilm for 1 h at 37°C with fluorescei-
nated avidin (avidin-FITC; Vector Laboratories) diluted
1:500 in 1% bovine serum albumin (BSA)/4× SSC
[instead of goat anti-biotin antibody (Vector Laborato-
ries)]. The slides were washed sequentially on a shaker
with 4× SSC, 0.1% Nonidet P-40 in 4× SSC, and 4×
SSC for 10 min each, and then rinsed with 2× SSC and
stained with 0.75 mg/ml propidium iodide for 3 min.
Microphotography and image capture
The chromosomal slides were observed under a Nikon
fluorescence microscope using Nikon filter sets B-2A
(450–490 nm) and ultraviolet (UV)-2A (330–380 nm).
Kodak Ektachrome American Standards Association
(ASA) 100 films were used for microphotography.
The digital FISH images were captured using the
550CW-QFISH application program of Leica Micro-
systems Imaging Solution Ltd. using a cooled CCD
camera (MicroMAX 782Y, Princeton Instruments)
mounted on a Leica DMRA microscope.
Results
Zoo-FISH analysis
We conducted the Zoo-FISH analysis on chromo-
somes from a male and a female specimen of T.
muenninki using all M. musculus chromosome
(MMU)-specific painting probes except for the Y
chromosome probe. Each chromosome of T. muen-
ninki could be identified on the basis of the Hoechst-
stained bands that were obtained by the replication
Sex-autosome fusions in Tokudaia muenninki 115

R-banding method (Fig. 1). The MMU probes for all
autosomes and the X chromosome were hybridized
successfully to T. muenninki chromosomes (TMU).
The images for the probes MMUX, MMU11, and
MMU16, are shown in Fig. 1a–c. The patterns for all
the T. muenninki chromosomes are summarized in
Fig. 1f. Although the long arm of the X chromosome
of T. muenninki was covered with the MMUX probe
Fig. 1 Karyotypic characterization in T. muenninki.a–cZoo-
FISH for T. muenninki with MMU-specific probes. Hybridization
of FITC- (green) labeled mouse painting probes, MMUX, to
PI-stained chromosomes of a T. muenninki female (a). Hybridiza-
tion of Cy3- (yellow) and Cy5- (pink) labeled mouse painting
probes, MMU11 (b)andMMU16(c), respectively, to Hoechst-
stained chromosomes of a T. muenninki male. Arrows and arrow-
heads indicate the hybridization signals on the sex chromosomes
and autosomes [TMU10 (b)andTMU15(c)], respectively. The
scale bar indicates 10 μm. (d,e) Mapping of T. muenninki chro-
mosomes by FISH using mouse genomic DNA clones of the 18S–
28S rRNA genes. Hybridization signals of the 18S–28S rRNA
genes were visualized by avidin-FITC on PI-stained chromosomes
(d). Hoechst G-banding patterns are shown in e.Arrows and
arrowheads indicate the hybridization signals on the sex chromo-
somes and a pair of autosomes (TMU1), respectively. The scale
bar indicates 10 μm. fComparative cytogenetic maps of T. muen-
ninki. The comparative cytogenetic maps showing chromosome
homologies between mouse and T. muenninki were constructed by
Zoo-FISH analysis with mouse probes. The numbers under the
chromosomes of T. muenninki indicate the chromosome numbers
for this species. The numbers inside the chromosomes indicate the
chromosome numbers for mouse that correspond to the chromo-
somal segments of T. muenninki indicated. Arrowheads indicate
the locations of the 18S–28S rRNA genes. Large heterochromatic
regions are shown in black
116 C. Murata et al.

(Fig. 1a), the short arms of the X and Y chromosomes
both hybridized with the mouse probes MMU11 and
MMU16 (Fig. 1b and c, respectively). The 18S–28S
ribosomal RNA genes were localized to the distal ends
of chromosome 1 and the short arms of the sex chro-
mosomes (Fig. 1d, e), which corresponded to MMU9
and MMU11, respectively (Fig. 1f).
Fourteen chromosomes of T. muenninki (TMU1, 2,
3, 5, 7, 9, 11, 14, 15, 17, 18, 19, 20, and 21) each were
hybridized with a single mouse probe; six chromo-
somes (TMU4, 6, 8, 10, 12, and 13) each hybridized
with two probes; and one chromosome (TMU16) was
hybridized with four probes (Fig. 1f). Thirty-four con-
served segments were detected between the mouse and
T. mu e n n i nki chromosomes. The hybridization pat-
terns between T. muenninki and the mouse probes
could be grouped into three categories: (1) 11 mouse
probes (MMU2, 3, 4, 6, 7, 9, 12, 14, 18, 19, and X)
each hybridized to a single chromosome or chromo-
somal segment of T. muenniki; (2) six mouse probes
(MMU1, 8, 11, 13, 15, and 16) each hybridized to two
chromosomes; and (3) three mouse probes (MMU5,
10, and 17) each hybridized to more than three chro-
mosomes (Fig. 1f).
CGH analysis
We performed CGH to identify the male-specific region
in the Y chromosome of T. muen n i n k i. CGH signals
with differently labeled genomic DNAs from a female
and a male were highlighted in the long arm of the Y
chromosome, as well as in the heterochromatic regions
of five autosomal pairs in both sexes (Fig. 2). The long
arm of the Y chromosome, Yq, was labeled intensely not
only by the male probe, which contained Y chromo-
somal DNA, but also by the female probe, although the
intensity of Hoechst fluorescence of Yq was not higher
than that of any of the other chromosomes (Fig. 2a–c).
The short arm of the Y chromosome, Yp, which is a
proto-autosomal segment that has been translocated to
the Y chromosome, as shown in Fig. 1, was stained
equally by the female- and male-derived probes
(Fig. 2d). Therefore, the loss or gain of a male-specific
chromosomal region was not detected in the T. muen -
ninki male by CGH analysis. Furthermore, when unla-
beled female DNA and mouse C0t-1 DNA were used in
the CGH analysis as competitors, no male-specific
region was detected in the T. muenninki male (data not
shown). In addition, the sex-specific region was not
Fig. 2 CGH images of a male (a–d) and a female (e–h) meta-
phase spread for T. muenninki. The Hoechst G-banding patterns
are shown in aand e. The CGH images (d,h) were obtained by
merging the images of metaphase spreads hybridized with
female genomic DNA (b,f) and male genomic DNA (c,g).
Arrows indicate the sex chromosomes. Scale bar represents
10 μm
Sex-autosome fusions in Tokudaia muenninki 117

detected in diploid mitotic complements of the T. mu e n -
ninki female (Fig. 2e–h).
Comparative FISH and FISH analysis
We mapped mouse cDNA clones of 22 X-linked genes
on chromosomes derived from a T. muenniki female
by direct R-banding FISH. All 22 genes were mapped
on the long arm of the X chromosome in T. muenninki.
The gene order was identical to that of the other two
Tokudaia species and the laboratory rat, R. norvegicus
(Fig. 3, Kobayashi et al. 2008). The hybridization
patterns of four genes on the X chromosome, ELK1
(Fig. 4a, b), SAT1 (Fig. 4c, d), SLC25A5 (Fig. 4e, f),
and PLXNA3 (Fig. 4g, h), are shown in Fig. 4.
Mapping analysis of the SRY gene could not be used
to identify the original Y chromosome region because
multiple copies of SRY are distributed over the entire Yq
and centromere region of the Y chromosome in T. muen-
ninki (Murata et al. 2010). In light of this, we used nine
mouse cDNA clones (unpublished data) to screen for
single copy genes on the Y chromosome by Southern
blot analysis. The DDX3Y and UTY genes appeared to
occur as a single copy in the male T. muenninki genome.
Thus, we cloned the T. muenninki cDNA for both
DDX3Y and UTY and used them as probes for FISH.
The sequence identity of the DDX3Y and UTY genes
between mouse and T. muenninki was 95%. Both the
DDX3Yand UTY genes were localized in T. muenninki to
the proximal region of the centromere on Yp (Fig. 4i–l).
Given that the MMU11 painting probe hybridized
to both Xp and Yp of T. muenninki, we performed
comparative FISH mapping using probes from mouse
cDNA clones of seven genes located on chromosome
11. All seven genes mapped to both Xp and Yp in T.
muenninki. The hybridization patterns of four genes,
STK10 (Fig. 5a–d), DOC2B (Fig. 5e–h), COASY
(Fig. 5i–l), and CBX2 (Fig. 5m–o), are shown in
Fig. 5. The gene orders on both Xp and Yp were iden-
tical to that on chromosome 11 of mouse (Fig. 3). How-
ever, CBX2 was also localized to chromosome 14 in
both sexes of T. muenniki (Fig. 5m–o).
Discussion
Ancestral karyotype of the genus Tokudaia
With the exception of several chromosome rearrange-
ments described below, the karyotype of T. muenninki
was largely identical to the one reported previously,
which was predicted from maps of T. osi m e n s i s and T.
Fig. 3 Comparative cytogenetic maps of seven genes from
mouse chromosome 11 on the sex chromosomes of T. muenninki
(TMUX, TMUY) and mouse chromosome 11 (MMU11) and 22
X-linked genes in T. muenninki and T. tokunoshimensis. The
locations and order of the genes are shown on the side of each
chromosome. The gene order and ideogram of MMU11 was
taken from the NCBI database (http://www.ncbi.nlm.nih.gov/
21/9/2011). The gene order and ideograms of T. tokunoshimen-
sis X chromosomes were taken from Kobayashi et al. (2008).
TTO, T. tokunoshimensis chromosome
118 C. Murata et al.

tokunoshimensis generated by chromosome painting
(Nakamura et al. 2007): MMU1b/17a/17e, a single seg-
ment of MMU14, and the two distinct chromosomal
segments of MMU15 occurred in T. muenn i n k i , whereas
MMU1b/17a, two distinct chromosomal segments of
MMU14, and a single segment of MMU15 occurred in
T. osi m e n s i s and T. tokunoshimensis. The former seg-
ments found in T. muenninki are also conserved in the
ancestral karyotype of the genus Apodemus (Matsubara
et al. 2004), which is the most closely related group to the
genus Tokudaia in Murinae (Michaux et al. 2002;Sato
and Suzuki 2004;Roweetal.2008), suggesting that the
ancestor of genus Tokudaia might have the same seg-
ments as T. muenninki, MMU1b/17a/17e, two distinct
chromosomal segments of MMU14 and a single segment
of MMU15. Therefore, we have demonstrated that the
ancestral karyotype of the genus Tokudaia had a diploid
chromosome number of 48 and contained the following
chromosomes that are homologous to mouse: 1a, 1b/17a/
17e, 2, 3, 4, 5a, 5b/11a, 5c/6, 7/19, 8a, 8b, 9, 10a, 10b/
17b, 10c/17c, 11b/16a, 12/17d, 13a, 13b/15a, 14, 15b,
16b, 18, X, and Y (Fig. 6). Lineage-specific chromosome
rearrangements in T. muenninki, inferred from the ances-
tral karyotype, were as follows: (1) fusion between seg-
ments homologous to MMU11b/16a and the ancestral Y
in TMUY; (2) centric fusions between segments homol-
ogous to MMU11b/16a and MMUX in TMUX and
between segments homologous to MMU10b/17b and
MMU13b/15a in TMU16; and (3) two pericentric inver-
sions in TMU17 and TMU19 (Fig. 6).
Furthermore, the localization of the 18S–28S ribo-
somal RNA genes to two chromosomal segments
homologous to MMU9 and MMU11 in T. muenninki
(Fig. 1d–f) was also conserved in the other two Tokudaia
Fig. 4 Chromosome mapping of four X-linked genes and two
Y-linked genes on T. muenninki chromosomes. The hybridization
signals are indicated by arrows. Four X-linked genes, ELK1 (a,b),
SAT1 (b,c), SLC25A5 (e,f), and PLXNA3 (g,h), were mapped from
the central to the distal part of T. muenninki Xq in the order shown.
TwoY-linkedgenes,DDX3Y (i,j)andUTY (k,l) were localized to
the proximal region of the centromere in T. mu e n nink i Yp . R - a nd
Hoechst G-banding patterns are shown in (a,c,e,g,i,k)and(b,d,f,
h,j,l), respectively. The scale bar indicates 10 μm
Sex-autosome fusions in Tokudaia muenninki 119

species (Arakawa et al. 2002;Nakamuraetal.2007)and
six Apodemus species (Matsubara et al. 2004), which
suggested that no chromosomal variation had occurred
in T. muen n i nk i in these regions.
Fusion of autosomes and the sex chromosomes
The short arms of the X and Y chromosomes of T.
muenninki were painted by the MMU11 and MMU16
probes, which indicated that fusions had occurred
between a pair of autosomes and the sex chromosomes.
This was not observed in the other two Tokudaia species,
where the chromosomal segments recognized by
MMU11 and MMU16 corresponded to chromosome 3q
in T. os i m e n s i s and chromosome 10 in T. tokunoshimen-
sis, respectively (Nakamura et al. 2007). The fusion of a
pair of autosomes with both the X and Y chromosomes is
a rare chromosomal rearrangement in mammals.
The only other reported cases are in: the giant
mole-rat (Fukomys mechowii), the African pygmy
mouse (Mus minutoides), and three bovine species
(Boselaphus tragocamelus,Tragelaphus spekei,and
Tragelaphus imberbis)(Deuveetal.2006;Veyrunes
et al. 2004,2010;Gallagheretal.1998;Rubeset
al. 2008). Species with X-autosome translocations
all have intercalary heterochromatic blocks (IHBs)
between the autosomal and the ancestral sexual
segments, which probably enables the timing of
Fig. 5 Chromosome mapping of the STK10 (a–d), DOC2B (e–h),
COASY (i–l), and CBX2 (m–o)genesonT. muenninki chromo-
somes. The STK10,DOC2B,andCOASY genes were localized to
both Xp (a,e,i) and Yp (c,g,k)inT. mu e n nink i .CBX2 was
mapped to Xp, Yp, and a pair of autosomes (TMU14) in the male
(m) and Xp and TMU14 in the female (o). The hybridization
signals are indicated by arrows on the sex chromosomes and
arrowheads on the autosomes. R- and Hoechst G-banding patterns
are shown in (a,c,e,g,i,k,m,o) and (b,d,f,h,j,l,n),
respectively. The scale bar represents 10 μm
120 C. Murata et al.

replicationofthesexandautosomalsegmentstobe
regulated independently (Dobigny et al. 2004). The
centromeric heterochromatin block of T. mue n n i n ki
might function as an IHB and an X-autosomal
boundary in this species (Murata et al. 2010).
Avoidance of Y chromosome loss through fusion
with an autosome
T. mu en n i nki is known to be the only species with a Y
chromosome in the genus Toku da i a ; as a consequence, it
follows the general XY pattern of mammalian sex
determination (Tsuchiya et al. 1989). However, our
results indicated that the Y chromosome of T. muenninki
has evolved in a unique manner. The Y chromosome
might have had instability persisting in the common
ancestral species of the genus Tok u d a i a (Fig. 7). In an
ancestral lineage common to the two XO/XO spiny rats,
T. osimensis and T. tokunoshimensis, most of the Y-
linked genes (except SRY and RBMY1A1)escapedto
the X chromosome, and the Y chromosome was lost
subsequently (Kuroiwa et al. 2010). In contrast, in the
ancestral population of T. muenni n k i , the X and Y
chromosomes fused with a pair of autosomes.
The enlargement of the Y chromosome through fusion
with an autosome extended the pseudoautosomal regions
(PARs). PARs behave like an autosomal pair and recom-
bine during male meiosis, which is thought to play a
critical role in spermatogenesis (Kauppi et al. 2011;
Matsuda et al. 1992b; Mohandas et al. 1992). The XY
male mice with inverted X PAR, Y PAR flanked at the
distal end by X PAR boundary together with adjacent
X-specific material, or both of the variant PARs, are
known to produce XO progeny by leading to the produc-
tion of unusual X and Y products and consequent sex
chromosome loss (Burgoyne and Evans 2000). If variant
PAR was associated with the Y chromosome loss in an
ancestral lineage common to the two XO/XO spiny rats,
T. os i m e n s i s and T. tokunoshimensis, the Y chromosome
would be stabilized by the acquisition of new PARs in the
ancestral lineage of T. muenninki, and thus, loss of the Y
chromosome was avoided. To test this hypothesis, future
research should include the identification and sequencing
of an ancestral PAR in three Tokudaia species.
Fig. 6 A schematic representation of the ancestral karyotype of the
Tokudaia species and chromosome rearrangements that occurred in
T. muenninki after its divergence from the common ancestor. The
numbers inside the chromosomes indicate the mouse chromosome
numbers that correspond to the indicated chromosomal segments of
the Tokudaia species. The lower chromosomes correspond to the
chromosomes of T. muenninki,andthenumbers underneath are the
chromosome numbers for this species. The numbers above the
upper chromosomes, those of the schematic ancestral karyotype,
indicate the chromosomes, chromosomal segments, or chromo-
some associations that are homologous to mouse chromosomes.
They were numbered following the nomenclature of Stanyon et al.
(2004) and Nakamura et al. (2007). Arrows show how the chro-
mosomes of T. muenninki were derived from the ancestral karyo-
type. Two-pronged arrows to a single chromosome indicate the
successive occurrence of fusions. “Inv”indicates a pericentric
inversion
Sex-autosome fusions in Tokudaia muenninki 121

Most human Y-linked genes are relics of a recent
autosomal addition (Waters et al. 2001). The PAR of
placental mammals was part of a larger autosomal
addition to the X and Y chromosomes 130–80 million
years ago (MYA) (Toder and Graves 1998), which
suggests that the ancestral PAR of placental mammals
was larger than the present human PAR (Waters et al.
2007). Enlargement of the Y chromosome by the
addition of an autosome could be an important chro-
mosomal phenomenon to stabilize X–Y pairing in the
lineage of placental mammals.
Structure of the giant X and Y chromosomes
All mouse cDNA clones of the X-linked genes were
mapped to Xq (Fig. 3). The gene order was consistent
with that of rat and the two other Tokudaia species
(Kobayashi et al 2008), which suggests that the proto-X
chromosome was retained in Xq and had not undergone
chromosomal rearrangement, e.g., inversion and deletion
on a broad scale.
In our CGH analysis, probes derived from either
males or females hybridized to Yq of T. muenninki
(Fig. 2). This might have been caused by the accumu-
lation of repetitive sequences on Yq that were common
to both sexes. The accumulation of numerous
repetitive sequences could be the reason why a male-
specific region was not detected clearly by CGH, even
though many SRY sequences are known to occur on
Yq of T. muenninki (Murata et al. 2010).
Previous attempts to map the SRY gene did not
detect an ancestral Y segment that was common to
other mammalian Y chromosomes due to the large
number of mainly nonfunctional SRY copies. In the
present study, using FISH to map two single-copy
Y-linked genes, DDX3Y and UTY, we identified the
region of Yp close to the centromere as the euchromatic
region of the proto-Y chromosome in the genus Toku-
daia (Fig. 4). Now that this region has been identi-
fied, further characterization of the Y-linked genes
in T. muenninki by Southern blotting and FISH
analyses is needed to reveal the gene content of
the original Y region. Sequence determination of
the region using a bacterial artificial chromosome
libraryisalsorequiredtodeterminethegeneorder
and gene structure of the putative ancestral Y
chromosome in T. mue n n i n k i .
Neo-X? Neo-Y?
Comparative FISH mapping of seven mouse cDNA
clones linked to chromosome 11 showed that the order
Fig. 7 The evolution of sex chromosomes, SRY, and CBX2 in
the genus Tokudaia.aThe evolutionary events inferred from the
present study (red) and previous studies (black) are shown in the
phylogeny, together with the geographical distribution of Toku-
daia species. T. muenninki,T. osimensis, and T. tokunoshimensis
inhabit only Okinawa-jima, Amami-Osima, and Tokunoshima
Islands, respectively. b,cAdult female (b) and juvenile to sub-
adult male (c)ofT. muenninki trapped as part of conservation
activities
122 C. Murata et al.

of genes on T. muenninki Xp and Yp was identical to that
of mouse chromosome 11. No obvious chromosome
rearrangement, e.g., inversion, was detected between
the X and Y chromosomes. However, it remains possi-
ble that some divergence in nucleotide sequence has
occurred between the X and Y chromosomes if the
proto-autosomal segments are in the early stages of
differentiation. On the basis of mitochondrial sequence
data for cytochrome b, divergence times between T.
muenninki and the two other species are estimated to
be around 2.5–2.7 MYA (Murata et al. 2010;onthe
basis of the substitution rate of this gene in murids, 4.8%
per MY; Suzuki et al. 2003). In the black muntjac
(Muntiacus crinifrons), some sequence divergence has
occurred between neo-sex chromosomes within the past
0.5 MY (Zhou et al. 2008). Therefore, there has been
sufficient time for mutations to accumulate between the
sex chromosomes of T. muenninki.
Duplication of CBX2 in the genus Tokudaia
Gene duplication has been proposed as a primary
source of material for the acquisition of evolutionary
novelties, e.g., new gene functions and new patterns of
expression (Ohno 1970; Sidow 1996). Several mech-
anisms have been put forward to explain how paralogs
diverge from one another. In one process, asymmetric
evolution, one gene copy retains the ancestral function
through purifying selection, whereas the other copy
can undergo neofunctionalization or pseudogenization
(Lynch and Conery 2000). Such a duplication event
allowed the evolution of a sex-determining gene in a
model fish species, medaka (Oryzas latipes, Schartl
2004).
Previously, we examined the copy numbers and chro-
mosomal locations of ten genes, including CBX2,to
identify candidate genes for sex determination in the
two Tokudaia species that lack SRY (Kuroiwa et al.
2011). The Cbx2/CBX2 gene is a single copy gene in
mice and humans and is known be involved in gonadal
differentiation. The knockout of Cbx2 in mouse causes
male-to-female sex reversal, and that mouse with the
karyotype 40, XY has normal female external genitalia,
which indicates that Cbx2/CBX2 represses ovarian
development (Katoh-Fukui et al. 1998; Biason-Lauber
et al. 2009). CBX2 occurs at two loci in T. osimensis and
T. tokunoshimensis, owing to the translocation of a
duplicated copy of the gene to another autosome. Fur-
thermore, two or three more copies of CBX2 are present
in males of these species as compared with females. It is
likely that these extra copies of CBX2 repress ovarian
development and hence cause the undifferentiated
gonad to develop testes in these species. Therefore, the
differences in gene dosage between sexes might
be involved in a novel sex-determining mechanism
(Kuroiwa et al. 2011). In the present study, we have
shown that T. muenninki also possesses multiple copies
of CBX2. This suggests that the duplication of CBX2
occurred in a common ancestor of all three Toku d a i a
species, after which the autosomal segment that
contained the original CBX2 copy became linked to
the T. muenninki sex chromosomes through chromo-
somal fusion. The comparison of the copy number of
CBX2 between T. muenninki male and female is needed
to reveal when Tokudaia species acquired the extra
copies of CBX2 in male genome.
T. muenninki possesses multiple copies of the SRY
gene on the Y chromosome (Murata et al. 2010). Most
of the copies are pseudogenes, with a complete ORF
conserved in only three copies (Murata et al. 2010). A
mutation, which results in a single amino acid substi-
tution in the DNA binding surface domain of the
HMG box of all SRY copies, might have weakened
the DNA binding ability of the SRY protein in T.
muenninki. If the Y-linked CBX2 in T. muenninki has
acquired a male-specific function through early differ-
entiation between Xp and Yp, CBX2 might also have
become an important gene for sex determination in T.
muenninki in the presence of a weakened SRY. Future
research should include the sequencing of each copy
of CBX2 and a functional analysis of the SRY genes in
T. muenninki to test this hypothesis.
Acknowledgments The authors thank C. Nishida-Umehara, K.
Matsubara, M. Tsuji. K. Sahara, and A. Yoshido for help with cell
culture and FISH. This work was supported by a Grant-in-Aid for
Scientific Research on Priority Areas from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and a grant
from the Naito Foundation, Japan.
References
Arakawa Y, Nishida-Umehara C, Matsuda Y, Sutou S, Suzuki H
(2002) X-chromosomal localization of mammalian Y-linked
genes in two XO species of the Ryukyu spiny rat. Cytogenet
Genome Res 99:303–309
Biason-Lauber A, Konrad D, Meyer M, Beaufort C, Schoenle
EJ (2009) Ovaries and female phenotype in a girl with 46,
Sex-autosome fusions in Tokudaia muenninki 123

XY karyotype and mutations in the CBX2 gene. Am J Hum
Genet 84:658–663
Burgoyne PS, Evans EP (2000) A high frequency of XO off-
spring from XPaf Y* male mice: evidence that the Paf
mutation involves an inversion spanning the X PAR
boundary. Cytogenet Cell Genet 91:57–61
Deuve JL, Bennett NC, O’Brien PCM et al (2006) Complex
evolution of X and Y autosomal translocations in the giant
mole-rat, Cryptomys mechowi (Bathyergidae). Chromo-
some Res 14:681–691
Dobigny G, Ozouf-Costaz C, Bonillo C, Volobouev V (2004)
Viability of X-autosome translocations in mammals: an
epigenomic hypothesis from a rodent case-study. Chromo-
soma 113:34–41
Elliott DJ (2004) The role of potential splicing factors including
RBMY, RBMX, hnRNPG-T and STAR proteins in sper-
matogenesis. Int J Androl 27:328–334
Gallagher DS, Davis SK, De Donato M et al (1998) A karyo-
typic analysis of nilgai, Boselaphus tragocamelus (Artio-
dactyla: Bovidae). Chromosome Res 6:505–513
Honda T, Suzuki H, Itoh M (1977) An unusual sex chromosome
constitution found in the Amami spinous country-rat, Toku-
daia osimensis osimensis. Jpn J Genet 52:247–249
Honda T, Suzuki H, Itoh M, Hayashi K (1978) Karyotypical
differences of the Amami spinous country-rats, Tokudaia
osimensis osimensis obtained from two neighbouring
islands. Jpn J Genet 53:297–299
Katoh-Fukui Y, Tsuchiya R, Shiroishi Tet al (1998) Male-to-female
sex reversal in M33 mutant mice. Nature 393:688–692
Kauppi L, Barchi M, Baudat F et al (2011) Distinct properties of
the XY pseudoautosomal region crucial for male meiosis.
Science 331:916–920
Kobayashi T, Yamada F, Hashimoto T et al (2007) Exceptional
minute sex-specific region in the X0 mammal, Ryukyu
spiny rat. Chromosome Res 15:175–187
Kobayashi T, Yamada F, Hashimoto T et al (2008) Centromere
repositioning in the X chromosome of XO/XO mammals,
Ryukyu spiny rat. Chromosome Res 16:587–593
Kominami R, Mishima Y, Urano Y, Sakai M, Muramatsu M (1982)
Cloning and determination of the transcription termination site
of ribosomal RNA gene of the mouse. Nucl Acids Res
10:1963–1979
Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge
R (1991) Male development of chromosomally female
mice transgenic for Sry. Nature 351:117–121
Kuramochi S, Matsuda Y, Okamoto M et al (1999) Molecular
cloning of the human gene STK10 encoding lymphocyte-
oriented kinase, and comparative chromosomal mapping of
the human, mouse, and rat homologues. Biochem Biophys
Res Commun 270:522–527
Kurohara K, Matsuda Y, Nagabukuro A, Tsuji A, Amagasa T,
Fujisawa-Sehara A (2000) Meltrin beta (ADAM19) gene:
cloning, mapping, and analysis of the regulatory region.
Immunogenetics 49:369–375
Kuroiwa A, Watanabe T, Hishigaki H, Takahashi E, Namikawa T,
Matsuda Y (1998) Comparative FISH mapping of mouse and
rat homologues of twenty-five human X-linked genes. Cytoge-
net Cell Genet 1:208–212
Kuroiwa A, Ishiguchi Y, Yamada F, Abe S, Matsuda Y (2010)
The process of a Y-loss event in an XO/XO mammal, the
Ryukyu spiny rat. Chromosoma 119:519–526
Kuroiwa A, Handa S, Nishiyama C et al (2011) Additional
copies of CBX2 in the genomes of males of mammals
lacking SRY,theAmamispinyrat(Tokudaia osimensis)and
the Tokunoshima spiny rat (Tokudaia tokunoshimensis).
Chromosome Res 19:635–644
Lynch M, Conery JS (2000) The evolutionary fate and conse-
quences of duplicate genes. Science 290:1151–1155
Matsubara K, Nishida-Umehara C, Tsuchiya K, Nukaya D,
Matsuda Y (2004) Karyotypic evolution of Apodemus
(Muridae, Rodentia) inferred from comparative FISH analyses.
Chromosome Res 12:383–395
Matsuda Y, Chapman VM (1995) Application of fluorescence in
situ hybridization in genome analysis of the mouse. Elec-
trophoresis 16:261–272
Matsuda Y, Harada Y-N, Natsuume-Sakai S et al (1992a) Loca-
tion of the mouse complement factor H gene (cfh) by FISH
analysis and replication R-banding. Cytogenet Cell Genet
61:282–285
Matsuda Y, Moens PB, Chapman VM (1992b) Deficiency of X
and Y chromosomal pairing at meiotic prophase in sper-
matocytes of sterile interspecific hybrids between labora-
tory mice (Mus domesticus) and Mus spretus. Chromosoma
101:483–492
Mazeyrat S, Saut N, Mattei MG, Mitchell MJ (1999) RBMY
evolved on the Y chromosome from a ubiquitously tran-
scribed X-Y identical gene. Nat Genet 22:224–226
Michaux JR, Chevret P, Filippucci MG, Macholan M (2002)
Phylogeny of the genus Apodemus with a special emphasis
on the subgenus Sylvaemus using the nuclear IRBP gene
and two mitochondrial markers: cytochrome band 12S
rRNA. Mol Phylogenet Evol 23:123–136
Mohandas TK, Speed RM, Passage MB et al (1992) Role of the
pseudoautosomal region in sex-chromosome pairing dur-
ing male meiosis: meiotic studies in a man with a deletion
of distal Xp. Am J Hum Genet 51:526–533
Murata C, Yamada F, Kawauchi N, Matsuda Y, Kuroiwa A
(2010) Multiple copies of SRY on the large Y chromosome
of the Okinawa spiny rat, Tokudaia muenninki. Chromo-
some Res 18:623–634
Nakamura T, Kuroiwa A, Nishida-Umehara C, Matsubara K,
Yamada F, Matsuda Y (2007) Comparative chromosome
painting map between two Ryukyu spiny rat species, Toku-
daia osimensis and Tokudaia tokunoshimensis (Muridae,
Rodentia). Chromosome Res 15:799–806
Ohno S (1970) Evolution by gene duplication. Springer, Berlin–
Heidelberg–New York
Rowe KC, Reno ML, Richmond DM, Adkins RM, Steppan SJ
(2008) Pliocene colonization and adaptive radiations in
Australia and New Guinea (Sahul): Multilocus systematics
of the old endemic rodents (Muroidea: Murinae). Mol
Phylogenet Evol 47:84–101
Rubes J, Kubickova S, Pagacova E et al (2008) Phylogenomic
study of spiral-horned antelope by cross-species chromo-
some painting. Chromosome Res 16:935–947
Sato JJ, Suzuki H (2004) Phylogenetic relationships and diver-
gence times of the genus Tokudaia within Murinae (Mur-
idae; Rodentia) inferred from the nucleotide sequences
encoding the Cytbgene, RAG1, and IRBP. Can J Zool
82:1343–1351
Schartl M (2004) Sex chromosome evolution in non-mammalian
vertebrates. Curr Opin Genet Dev 14:634–641
124 C. Murata et al.

Sidow A (1996) Gen(om ) e duplications in the evolution of
early vertebrates. Curr Opin Genet Dev 6:715–722
Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith
MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow
PN (1990) A gene from the human sex-determining region
encodes a protein with homology to a conserved DNA-
binding motif. Nature 346:240–244
Soullier S, Hanni C, Catzeflis F, Berta P, Laudet V (1998) Male sex
determination in the spiny rat Tokudaia osimensis (Rodentia:
Muridae) is not Sry dependent. Mamm Genome 9:590–592
Stanyon R, Yang F, Morescalchi AM, Galleni L (2004) Chro-
mosome painting in the long-tailed field mouse provides
insights into the ancestral murid karyotype. Cytogenet
Genome Res 105:406–411
Sutou S, Mitsui Y, Tsuchiya K (2001) Sex determination with-
out the Y chromosome in two Japanese rodents Tokudaia
osimensis osimensis and Tokudaia osimensis spp. Mamm
Genome 12:17–21
Suzuki H, Sato JJ, Tsuchiya K et al (2003) Molecular phylogeny
of wood mice (Apodemus, Muridae) in East Asia. Biol J
Linn Soc 80:469–481
Toder R, Graves JA (1998) CSF2RA,ANT3, and STS are auto-
somal in marsupials: implications for the origin of the
pseudoautosomal region of mammalian sex chromosomes.
Mamm Genome 9:373–376
Tsuchiya K, Wakana S, Suzuki H, Hattori S, Hayashi Y (1989)
Taxonomic study of Tokudaia (Rodentia: Muridae): I. Genetic
differentiation. Memoirs Nat Sci Museum. Tokyo 22:227–234
Veyrunes F, Catalan J, Sicard B et al (2004) Autosome and sex
chromosome diversity among the African pygmy mice,
subgenus Nannomys (Murinae; Mus). Chromosome Res
12:369–382
Veyrunes F, Catalan J, Tatard C et al (2010) Mitochondrial and
chromosomal insights into karyotypic evolution of the
pygmy mouse, Mus minutoides, in South Africa. Chromo-
some Res 18:563–574
Waters PD, Duffy B, Frost CJ, Delbridge ML, Graves JA (2001)
The human Y chromosome derives largely from a single
autosomal region added to the sex chromosomes 80–130
million years ago. Cytogenet Cell Genet 92:74–79
Waters PD, Wallis MC, Marshall Graves JA (2007) Mammalian
sex—origin and evolution of the Y chromosome and SRY.
Semin Cell Dev Biol 18:389–400
Yamada F, Kawauchi N, Nakata K et al (2010) Rediscovery after
thirty years since the last capture of the critically endangered
Okinawa spiny rat Tokudaia muenninki in the northern part of
Okinawa Island. Mammal Study 35:243–255
Zhou Q, Wang J, Huang L et al (2008) Neo-sex chromosomes in
the black muntjac recapitulate incipient evolution of mam-
malian sex chromosomes. Genome Biol 9:R98
Sex-autosome fusions in Tokudaia muenninki 125