Unequal Rates of Y Chromosome Gene Divergence during Speciation of the
Family Ursidae
Shigeki Nakagome,*1 Jill Pecon-Slattery, and Ryuichi Masuda*à
*Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Japan; Laboratory of Genomic
Diversity, National Cancer Institute-Frederick, Frederick, MD; and àDepartment of Genome Dynamics, Creative Research Initiative
‘‘Sousei’’, Hokkaido University, Sapporo, Japan
Evolution of the bear family Ursidae is well investigated in terms of morphological, paleontological, and genetic features.
However, several phylogenetic ambiguities occur within the subfamily Ursinae (the family Ursidae excluding the giant
panda and spectacled bear), which may correlate with behavioral traits of female philopatry and male-biased dispersal
which form the basis of the observed matriarchal population structure in these species. In the process of bear evolution,
we investigate the premise that such behavioral traits may be reflected in patterns of variation among genes with different
modes of inheritance: matrilineal mitochondrial DNA (mtDNA), patrilineal Y chromosome, biparentally inherited
autosomes, and the X chromosome. In the present study, we sequenced 3 Y-linked genes (3,453 bp) and 4 X-linked
genes (4,960 bp) and reanalyzed previously published sequences from autosome genes (2,347 bp) in ursid species to
investigate differences in evolutionary rates associated with patterns of inheritance. The results describe topological
incongruence between sex-linked genes and autosome genes and between nuclear DNA and mtDNA. In more ancestral
branches within the bear phylogeny, Y-linked genes evolved faster than autosome and X-linked genes, consistent with
expectations based on male-driven evolution. However, this pattern changes among branches leading to each species
within the lineage of Ursinae whereby the evolutionary rates of Y-linked genes have fewer than expected substitutions.
This inconsistency between more recent nodes of the bear phylogeny with more ancestral nodes may reflect the
influences of sex-biased dispersal as well as molecular evolutionary characteristics of the Y chromosome, and stochastic
events in species natural history, and phylogeography unique to ursine bears.
Introduction
Species within the bear family Ursidae include the
world’s largest carnivores and are distributed widely in
Eurasia and North and South America. Ursidae consists of
8 species: the spectacled bear (Tremarctos ornatus), sloth
bear (Ursus ursinus), sun bear (Ursus malayanus), Asiatic
black bear (Ursus thibetanus), American black bear (Ursus
americanus), brown bear (Ursus arctos), polar bear (Ursus
maritimus), and giant panda (Ailuropoda melanoleuca). The
progenitor of extant bears arose approximately 37–40 MYA,
followed by A. melanoleuca around 12 MYA, T. ornatus at
5–7 MYA and the progenitor of the subfamily Ursinae originating 4–6 MYA as indicated by the fossil record (Wayne
et al. 1991). This general pattern of Ursidae speciation is corroborated by genomic data such as chromosome karyology
(Nash and O’Brien 1987; Nash et al. 1998), protein electrophoresis (Slattery and O’Brien 1995), and molecular evolutionary studies (Talbot and Shields 1996b; Waits et al. 1999;
Yu et al. 2004, 2007; Pages et al. 2007).
Relationships between the 6 remaining species of the
subfamily Ursinae differ between full-length mitochondrial
DNA (mtDNA) (Yu et al. 2007) and nuclear genomic
markers. For example, mtDNA phylogenetic trees depict
U. ursinus diverging first within Ursinae 4–6 MYA, followed by a bifurcation forming 2 clades: one leading to
the ancestor of U. arctos and U. maritimus. The second
clade clearly defined the 2 species of black bear (U. amricanus and U. thibetanus) as sister taxa: a result observed
with autosome DNA (Yu et al. 2004), as well as other
1
Present address: Department of Integrated Biosciences, Graduate
School of Frontier Sciences, University of Tokyo, Kashiwa, Japan.
Key words: Ursidae, sex-linked genes, male-biased dispersal, female
philopatry, matriarchal structure.
E-mail: [email protected].
Mol. Biol. Evol. 25(7):1344–1356. 2008
doi:10.1093/molbev/msn086
Advance Access publication April 9, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
mtDNA genes (Talbot and Shields 1996a, 1996b; Waits
et al. 1999) and morphological studies (Kurten 1964;
Mazza and Rustioni 1994). Further, the placement of
U. malayanus within Ursinae remained ambiguous among
these previously published studies. These discordant phylogenetic results underscore the inherent difficulties in conducting evolutionary studies of species with rapid recent
evolution and provide compelling support for the continued
investigation of informative genomic markers. Here we assess the performance of 3 Y-linked (3,453 bp) and 4 Xlinked (4,960 bp) genes and conduct comparisons with mitochondrial and autosome genes to investigate differences
in evolutionary rates associated with mode of inheritance.
Genes on mammalian sex chromosomes evolve differently due to patterns of inheritance. The Y chromosome is
exclusively patrilineal, whereas the mode of inheritance for
X chromosome is one-third in males and two-third in females (Miyata et al. 1987). With the exception of the small
pseudoautosomal region, that is, 5% of the human Y chromosome (Rappold 1993), the remainder, termed the nonrecombining region of the Y chromosome (NRY), does not
undergo conventional recombination during male meiosis.
Consequently, genes in the NRY are thought to be under
strong selection for male-specific function or undergo degradation due to an accumulation of deleterious mutations
through Muller’s ratchet (Charlesworth B and Charlesworth D 1997), genetic hitchhiking (Charlesworth 1996),
background selection (Charlesworth 1996), and insertion
of retroposable elements (Charlesworth 1991). Most studies
of X–Y homologs indicate that genes on the Y chromosome
evolve faster than those on the X chromosome in primates,
carnivores, perissodactyls, and rodents (Haldane 1947;
Huang et al. 1997; Pecon-Slattery and O’Brien 1998;
Makova and Li 2002; Wolfe and Li 2003; Sandstedt and
Tucker 2005; Goetting-Minesky and Makova 2006) consistent with expectations of male-driven evolution (Haldane
1947; Miyata et al. 1987; Shimmin et al. 1993; Makova
and Li 2002; Goetting-Minesky and Makova 2006).
AB294728
AB292078
AB261838
AB261830
AB292070
AB261822
AB261814
AB294721
AB294723
AB294724
AB294725
AB294726
AB294727
AB292072
AB292073
AB292074
AB292075
AB292076
AB292077
AB261832
AB261833
AB261834
AB261835
AB261836
AB261837
AB261824
AB261825
AB261826
AB261827
AB261828
AB261829
AB292064
AB292065
AB292066
AB292067
AB292068
AB292069
AB261816
AB261817
AB261818
AB261819
AB261820
AB261821
AB261808
AB261809
AB261810
AB261811
AB261812
AB261813
AB292071
AB261831
AB261823
One male sample was used for each species.
Giant panda
Fibroblasts
a
Ailuropoda melanoleuca
Blood
Hair roots
Muscle
Hair roots
Liver
Hair roots and blood
Polar bear
American black bear
Asiatic black bear
Sun bear
Sloth bear
Spectacled bear
SMCX
SMCY
SRY
AB292063
AB261815
AB261807
Brown bear
Liver
Hokkaido Institute of Environmental
Science, Japan
Tennoji Zoo, Japan
Ikeda Zoo, Japan
Hunting, Honshu, Japan
Ueno Zoological Gardens, Japan
Kobe Municipal Oji Zoo, Japan
Tennoji Zoo and Yokohama Zoo,
Japan
Ueno Zoological Gardens, Japan
Ursus maritimus
Ursus americanus
Ursus thibetanus
Ursus malayanus
Ursus ursinus
Tremarctos ornatus
Polymerase chain reaction (PCR) primers were designed for the final intron of ZFY/X, the fourth intron of
SMCY/X, the third intron of PLP, the seventh intron of
ALAS2, and the coding and noncoding regions of SRY
Ursus arctos
Primer Design and Polymerase Chain Reaction Analysis
of 3 Y Chromosome Genes and 4 X Chromosome Genes
Species Identification
Tissue samples were obtained from male individuals
from 8 species of the family Ursidae (table 1). Total
DNA was extracted using the DNeasy tissue kit (Qiagen,
Tokyo, Japan) or the QIAamp DNA Micro kit (Qiagen).
Additional samples were included from female individuals
of the brown bear (U. arctos) and Asiatic black bear
(U. thibetanus) to confirm male specificity of primers developed for Y chromosome genes.
Table 1
Profiles of Samples Used in This Study
Materials and Methods
DNA Specimens
Common Name
Tissue Profilea
Source of Sample
ZFY
ZFX
GenBank Accession Number for Gene Sequence
PLP
ALAS2
Like many mammalian species (see Lawson Handley
and Perrin 2007), Ursinae species exhibit behavioral traits
of male dispersal and female philopatry. Ecological studies
demonstrate that North American (McLellan and Hovey
2001), European (Stoen et al. 2006), and Russian (Kojola
et al. 2003) populations of brown bears (U. arctos) exhibit
male bias in dispersal from the natal range, and thus, brown
bears are spatially structured in matrilineal assemblages
(Stoen et al. 2005). Similarly, the home range of males
is expanded by an order of magnitude compared with females of American black bears (U. americanus) (Nowak
1999), consistent with sex-biased dispersal and matriarchal
social structure (Onorato et al. 2004). In addition, these behavioral traits may be reflected in population genetic analyses of these species. For example, mtDNA haplotypes of
brown bears (U. arctos) on the Hokkaido Island of Japan
were separated clearly into 3 allopatric groups (Matsuhashi
et al. 1999), indicative of female philopatry. Thus, the
genetic consequences of sex-linked dispersal might be detected through comparisons of the level of genetic diversity
between patrilineal markers on the Y chromosome and matrilineal mtDNA (Seielstad et al. 1998; Oota et al. 2001;
Eriksson et al. 2006; Hammond et al. 2006).
In general, studies designed to assess male dispersal
and female philopatry are complex using models of population genetics, evolutionary genetics, and field observations. Herein, we employ a phylogenetic approach using
intron sequences of sex-linked, single-copy Y–X homologs
ZFY/X (zinc finger protein on Y/X), SMCY/X (selected
mouse cDNA on Y/X), along with X-linked PLP (proteolipid protein) and ALAS2 (aminolevulinate, delta-, synthase
2), and coding and adjacent noncoding regions of SRY
(sex-determining region on Y) to propose that evolution
of extant bears is likely associated with ongoing and historic sex-linked social behaviors. Notably, low substitution
rates in Y chromosome genes within more recent Ursinae
lineages of the bear phylogeny compared with those within
more ancestral branches may reflect the influence of malebiased dispersal.
AB294722
Sex Chromosome Evolution in Bears 1345
1346 Nakagome et al.
(supplementary table 1, Supplementary Material online). For
ZFY/X, SMCY/X, PLP, and ALAS2, the PCR primers were
designed from conserved exon sequences flanking the intron
regions, which were obtained by alignments of published
cDNA sequences for the human and mouse (GenBank accession numbers of the National Center for Biotechnology
Information : ZFY/X, NM_003411/NM_003410 in the human and NM_009570/NM_011768 in the mouse; SMCY/X,
NM_004653/NM_004187 in the human and NM_011419/
NM_0137668inthemouse;PLP,NM_199478.1inthehuman
and NM_011123.2 in the mouse; and ALAS2, NM_000032.2
in the human and NM_009653.2 in the mouse).
All PCR conditions and primer sets used in this study
were described in supplementary table 2 (Supplementary
Material online). Except for the volumes of rTaq DNA
polymerase (Takara Bio Inc., Otsu, Japan), all PCR mixtures were same as a total volume of 50 ll including 2 ll
of the DNA extract (50–100 ng/ll), 10 mM Tris–HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphate, and 0.25 lM of each prime. The volumes of rTaq DNA polymerase were used in 1.25 or 2.5
units. For ZFY/X and SMCY/X, the intron regions of Y
and X chromosomes were simultaneously amplified using
primers located on the exon regions (U-ZF-1F/1R and USMC-2F/2R). For ZFY/X, it was necessary to perform
nested PCR using 5 ll of first-round PCR products in
the primer set of U-ZF-2F/2R because nonspecific PCR
products where obtained in first-round PCR. In addition,
the PCR products of ZFY/X and SMCY/X for the brown bear
(U. arctos) and giant panda (A. melanoleuca) were cloned
using the TA cloning kit (Invitrogen, Carlsbad, CA) to design internal primers specific to each intron region of ZFY/X
or SMCY/X, respectively (supplementary table 1, Supplementary Material online). Using the combinations of the
exonic and internal primers, the intron regions of ZFY/X
and SMCY/X were separated into 2 segments to amplify
the specific region of Y or X chromosome, respectively
(supplementary table 2, Supplementary Material online).
For PLP and ALAS2, the amplification of intron regions
succeeded using primer sets situated in flanking exon regions
(supplementary table 2, Supplementary Material online). In
order to design the sequencing primers, the TA cloning was
conducted using the PCR products of the brown bear (U. arctos) and giant panda (A. melanoleuca) (supplementary table 3,
Supplementary Material online).
For SRY, the nucleotide sequence data of the brown bear
(U. arctos) (GenBank accession number: AY424666) were
used to design 2 primer pairs for seminested PCR; U-SRY1F/1R and 2F/2R (supplementary tables 1 and 2, Supplementary Material online). In the spectacled bear (T. ornatus),
seminested PCR was performed using U-SRY-1F/2R or
U-SRY-2F/1R (supplementary table 2, Supplementary Material online).
The male specificity of Y-linked gene primers was confirmed by the presence of PCR product in males and the absence in females of both the brown bear (U. arctos) and
Asiatic black bear (U. thibetanus). All PCR products were
purified with the QIAquick purification kit (Qiagen). PCR
sequencing reactions used the Thermo Sequenase Primer
Cycle Sequencing Kit (Amersham, Piscataway, NJ). Sequencing primers (supplementary table 3, Supplementary
Material online) newly designed in this study were 5# labeled with Texas red. The cycle PCR products were sequenced with an automated sequencer HITACHI SQ-5500.
Phylogenetic Analysis
Nucleotide sequences were compiled and aligned by
GeneWorks (IntelliGenetics, Inc., Mountain View, CA).
For phylogenetic analyses, gaps (insertion/deletions [indels]) and short interspersed elements (SINEs) were removed. For comparison with sex chromosome DNA data
sets of the present study, previously published sequences
of autosome genes TTR-intron 1 and IRBP-exon 1 (Yu
et al. 2004) were included (GenBank accession numbers
of previously published sequences are shown in supplementary table 4, Supplementary Material online).
Phylogenetically informative sites and variable sites
were obtained using MEGA version 3.1 (Kumar et al.
2004). Phylogenetic trees were constructed using
PAUP4.0b10 (Swofford 2002)under 3 different optimality criteria: minimum evolution (ME), maximum parsimony (MP),
and maximum likelihood (ML) methods. Under the ME criterion, the phylogenetic trees were reconstructed using the Tajima–Nei distance model (Tajima and Nei 1984). For the ML
analysis, hierarchical likelihood ratio tests were initially
introduced to compare the goodness of fit for 56 nucleotide
substitution models using Modeltest3.7 (Posada and Crandall
1998) for 3 individual data sets of Y-linked, X-linked, or
autosome genes, and additional analyses of combined
nuclear DNA data (Y-linked, X-linked, and autosome gene
regions) to determine the specific model parameters. ML
substitution models showed 1) the Hasegawa, Kishino, and
Yano (HKY) model with estimated nucleotide base frequencies of A 5 0.3004, C 5 0.1730, G 5 0.2044, and
T 5 0.3222; a transition:transversion ratio 5 1.7417 for
Y-linked genes; 2) the HKY þ gamma model with estimated
nucleotide base frequencies of A 5 0.3054, C 5 0.1919,
G 5 0.2239, and T 5 0.2788; a transition:transversion ratio
5 2.1345; gamma 5 0.0139 for X-linked genes; 3) the HKY
þ gamma model with estimated nucleotide base frequencies of
A 5 0.2208, C 5 0.2867, G 5 0.2692, and T 5 0.2233;
a transition:transversion ratio 5 3.021; gamma 5 0.006 forautosome genes; 4) HKY þ gamma model with estimated nucleotide base frequencies of A 5 0.2836, C 5 0.2084,
G 5 0.2281, and T 5 0.2800; a transition:transversion
ratio 5 2.1748; gamma 5 0.0171 for the combined nuclear
data set. In the ME, MP, and ML methods, optimal
trees were determined by an exhaustive search. The reliability
ofderivedphylogeneticrelationshipswasevaluatedusingbootstrap analyses, and clades supported by greater than 50% of
node bootstrap values were retained. For the ME and MP analyses,1,000iterationsofbootstrapwereperformedwithheuristic
tree searches employing the tree-bisection-reconnection
(TBR) branch swapping. For the ML bootstrapping, 100
iterations were implemented using these conditions.
A fourth method utilizing a Bayesian approach for
computing clade credibility values for nodes within the tree
was performed using program MrBayes (version 3.1.2)
(Huelsenbeck and Ronquist 2001). The Bayesian analysis
was also used in 3 individual data sets of Y-linked,
Sex Chromosome Evolution in Bears 1347
X-linked, or autosome genes and combined nuclear data
set. The nucleotide evolution models used in ML analyses
were also incorporated in Bayesian methods. Specific
parameters included: a random starting tree, no phylogenetic constraints, 4 Markov chains run for 2,000,000
generations, empirical estimates of stability-likelihood values set to burn-in at 10,000 generations, and tree sampling
every 100 generations. Two runs were performed to confirm the stability of posterior probability. The convergence
of Markov chains was assessed from the average standard
deviation (,0.01) and the potential scale reduction factor
(close to 1.000), as well as the log likelihood values.
Comparison of Substitution Rates
Differences in rates of substitution between nuclear
genes located on different chromosome regions were estimated by computing the pairwise genetic distances between
species. Using a constraint tree generated by phylogenetic
analyses of the combined data set of sex-linked and autosome genes (9,515 bp), branch lengths for each separate
gene category (Y-linked, X-linked, or autosome genes)
were computed using the method described in Sandstedt
and Tucker (2005) and expressed as the number of substitution per 100 sites. The substitution rates among nuclear
genes were compared as follows: the branch of node A to
the spectacled bear (T. ornatus), that of node A to 6 ursine
species, that of node A to node B, and that of node B to 6
ursine species (node A and node B as shown in fig. 3). Tests
for significant differences of substitution rates among Ylinked, X-linked, and autosome genes using STATISTICA
version 06J (StatSoft, Tokyo, Japan) adopted 1-way analysis of variance (Snedecor and Cochran 1989) and the post
hoc multiple comparison tests (Fisher’s protected leastsignificant difference [PLSD]). Significance levels of
P , 0.05 and P , 0.01 were adopted throughout.
Results
Portions of 3 Y chromosome genes and 4 X chromosome genes were sequenced in all 8 extant species of bears.
These regions included 1,090 bp of the ZFY-final intron,
1,096 bp of the SMCY-intron 4, and 1,267 bp of the SRY (coding and noncoding regions) for Y chromosome genes; as well
as 861 bp of the ZFX-final intron, 531 bp of the SMCX-intron
4, 1,004 bp of the PLP-intron 3, and 2,564 bp of ALAS2intron 7 for X chromosome genes (alignments shown in supplementary figs. 1–7, Supplementary Material online).
Repetitive elements were present within sex-linked
gene sequences in bears. Within the ZFY-final intron, a
SINE insertion occurred at nucleotide (nt) sites 366–540
shared by all 8 bear species (supplementary fig. 1, Supplementary Material online). Shared by 2 species (U. arctos
and U. maritimus), an identical SINE insertion was located
at nt 610–824 in the SMCY-intron 4 that differed only in
total lengths of the poly-A tail (14 and 12 bp for U. arctos
and U. maritimus, respectively, in supplementary fig. 2,
Supplementary Material online). In ALAS2-intron 7, an insertion occurred in A. melanoleuca and T. ornatus at
nt 938–1,698 and differed by 12 variable sites between
the 2 species (supplementary fig. 7, Supplementary Material
online).
Autoapomorphic indels occurred within these genes
unique to a given species. A dinucleotide repeat (AT)n varied between U. americanus and other species at nt 506–514
of the SMCY-intron 4 (supplementary fig. 2, Supplementary
Material online). A 21-bp insertion was founded at nt
343–363 in the ZFY-final intron of T. ornatus and an 11-bp
insertion at nt 565–575 in A. melanoleuca (supplementary fig. 1, Supplementary Material online). Tandem repeats
of TTGA motif at nt 604–615 in the ZFX-final intron varied
from 5 repeats in T. ornatus, 3 in U. thibetanus, and 4 in the
other species (supplementary fig. 4, Supplementary Material
online). In X-linked genes, a microsatellite (CA)n locus occurredatnt2,337–2,398intheALAS2-intron7(supplementary
fig. 7, Supplementary Material online) and varied among bear
species as the following: U. arctos (14 repeats), U. maritimus
(20 repeats), U. americanus (25 repeats), U. thibetanus
(26 repeats), U. malayanus (19 repeats), U. ursinus (25
repeats), T. ornatus (20 repeats), and A. melanoleuca
(11 repeats).
The sequence for SRY consisted of the adjacent 5#
noncoding flank (nt 1–434), the SRY gene (a single exon
nt 435–1,100), and the 3# noncoding flank (nt 1,101–
1,267) (supplementary fig. 3, Supplementary Material online). In U. arctos, there was 1 missense mutation at nt
1,098 (T to C), which extended the SRY protein by an additional 11 amino acids (33 bp) relative to the other species.
Phylogenetic Analyses
The reconstructions of bear phylogeny were performed on sequences in which indels and SINEs were removed. The resultant alignments consisted of 1,033 bp for
ZFY, 848 bp for SMCY, 1,266 bp for SRY, 850 bp for ZFX,
520 bp for SMCX, 993 bp for PLP, and 1,720 bp for ALAS2.
The number and the percentage of variable sites, parsimoniously informative sites, and the consistency index obtained from MP analysis were determined (table 2). In
addition, the distribution of indels being parsimoniously informative was estimated as diagnostic sites for the contribution to each bear lineage defined in the sex-linked and
autosome trees (table 3 and supplementary table 5, Supplementary Material online). Among nuclear genes, Y-linked
genes have the highest numbers of substitutions and more
diversity than other genes. Despite of the largest partitions
of sequences, X-linked genes show the lowest numbers of
substitutions. Nuclear genes had very little homoplasy, with
Y-linked genes having the highest consistency index 5
0.99 of all genes categories. On average, Y-linked genes
were the most parsimonious informative, followed by autosome genes, with X-linked genes being the most conserved for nuclear sequence data (table 2).
Phylogenetic trees were reconstructed with concatenated sequences of ZFY-SMCY-SRY for Y chromosome
genes (fig. 1A) and of ZFX-SMCX-PLP-ALAS2 for X chromosome genes (fig. 1B) using A. melanoleuca as an outgroup. Both trees depicted the early divergence of
T. ornatus and strongly supported the monophyly of the
1348 Nakagome et al.
Table 2
Phylogenetic Parameters of Y Chromosome and X Chromosome Genes in Ursidae
Gene
Regions
Y chromosome
genes
X chromosome
genes
Autosome genes
a
b
Gene
Segments
ZFY-final intron
SMCY-intron 4
SRY 5#-noncoding
region
SRY 3#-noncoding
region
SRY-coding region
Combined
ZFY-final intron
SMCY-intron 4
PLP-intron 3
ALAS2-intron 7
Combined
IRBP-exon 1
TTR-intron 1
Combined
Sequence
Length (bp)
No. of
Variable
Sites
% of
Variable
Sites
No. of
Parsimonious
Informative
Sites
% of
Parsimonious
Informative
Sites
60
50
14
5.50
4.56
3.23
10
6
3
0.92
0.55
0.69
8
4.79
1
0.60
34
166
21
23
31
53
128
37
55
92
4.86
4.76
2.44
4.33
3.09
2.07
2.58
2.89
5.15
3.92
3
23
2
4
6
3
15
4
15
19
0.43
0.66
0.23
0.75
0.60
0.12
0.30
0.31
1.41
0.81
1,090
881 (1,094–1,096)a
434
167
666
3,453
861
531
1,004
1,803 (2,562–2,564)b
4,960
1,280
1,067
2,347
Consistency
Index
0.994
0.949
0.945
1,094 bp, the polor bear with SINE insertion; 1,096 bp, the brown bear with the SINE insertion.
2,562 bp, the giant panda with 759 insertion; 2,564 bp, the spectacled bear with 761-bp insertion.
6 ursine species that subsequently split to form 2 lineages:
one composed of U. arctos, U. maritimus, and U. americanus and the other consisting of a less resolved clade of
U. ursinus, U. malayanus, and U. thibetanus. Both Y
and X chromosome phylogenetic trees, and the shared
SINE insertion of SMCY-intron 4, supported the common
ancestry of U. arctos and U. maritimus. The divergence of
U. ursinus, U. malayanus, and U. thibetanus existed as a trichotomy with X-linked genes and was weakly supported by
only the ME analyses of Y-linked genes.
Our reanalysis of previously published autosome
genes of TTR-intron 1 and IRBP-exon 1 recapitulated the
original findings (Talbot and Shields 1996b; Waits et al.
1999; Slattery et al. 2000; Yu et al. 2004, 2007). The phylogenetic trees (fig. 2) indicated a close association between
U. arctos and U. maritimus observed with sex-linked genes
but differ by uniting U. americanus with U. thibetanus. Autosome genes further supported the clade of U. malayanus
and U. ursinus as sister taxa. Thus, the phylogeny based on
autosome genes defined 2 clades that separated the 6 ursine
species into a lineage composed of the 2 species U. malayanus and U. ursinus and with the remainder of U. arctos, U.
maritimus, U. americanus, and U. thibetanus formed the
second lineage (fig. 2).
A combined analysis of concatenated nuclear genes
(9,511 bp) resulted in a well-supported consensus phylogeny for bear speciation (fig. 3). The early divergence of T.
ornatus and the monophyly of Ursinae were recovered,
consistent with results of the separate analyses of sex-linked
(fig. 1A and B) and autosome genes (fig. 2). Within Ursinae,
2 lineages were established, which is consistent with the
phylogeny of autosome genes (fig. 2). The first was composed of Asian species of U. malayanus and U. ursinus as
sister species. The second clade, composed of the remaining
4 Ursinae positioned both species of black bears (U. americanus and U. thibetanus) as more basal to the sister taxa of
U. arctos and U. maritimus.
Based on lineages within Ursinae defined in each phylogeny of Y-linked, X-linked (fig. 1A and B), or autosome
genes (fig. 2), we examined the distribution of diagnostic
sites for the lineages and investigated the contribution of
each chromosomal gene to the specific topology of combined
nuclear phylogeny (table 3 and supplementary table 5,
Supplementary Material online). The monophyly of Ursinae was strongly supported in all genes, especially Y
chromosome genes (N 5 18). For the first divergence of
6 species into 2 Asian species and the other (fig. 3), the diagnostic sites were shown only in autosome genes (N 5 2
in the former lineage and N 5 4 in the latter lineage). On
the other hand, the clade of U. arctos, U. maritimus, and
U. americanus was specific to sex-linked genes (N 5 5 in Ylinked genes and N 5 4 in X-linked genes). Weak support
for the linage of U. thibetanus, U. ursinus, and U. malayanus or U. thibetanus and U. americanus was obtained in
both X-linked (N 5 2) and autosome genes (N 5 1) or only autosome genes (N 5 1). The monophyly of U. arctos
and U. maritimus was supported consistently across all nuclear genes within Ursinae (N 5 3 in Y-linked, N 5 2 in
X-linked, and N 5 2 in autosome genes). Overall, nodal
support within the combined nuclear phylogeny varied
from moderate to high and the pattern of substitution
was diagnostic and parsimoniously informative (consistency index 5 0.96) and exhibited little homoplasy.
Comparison of Rates of Substitution among Genome
Regions
Nucleotide substitution rates were compared between
Y-linked, X-linked, and autosome genes using the topology
derived from the combined data analysis as a constraint tree
to compute genetic distances estimated from branch lengths
(table 4). Overall, the substitution rate of Y chromosome
genes was the highest, intermediate for autosome genes,
Table 3
The Distribution of Diagnostic Sites Located in Y-Linked, X-Linked, and Autosome Genes That Define Each of the Bear Lineages
Y Chromosome Genes
Lineages
Monophyly of Ursus
arctos and Ursus
maritimus
Monophyly of Ursus
ursinus and Ursus
malayanus
Monophyly of Ursus
thibetanus and Ursus
americanus
U. americanus,
U. maritimus, and
U. arctos group
U. thibetanus, U.
ursinus, and U.
malayanus group
U. arctos, U.
maritimus, U.
amcericanus, and
U. thibetanus
SMCY-Intron
4
SRY
Y Chromosome
ZFX-Final
Intron
SMCX-Intron
4
Autosome Genes
PLP-Intron
3
ALAS2-Intron
7
X Chromosome
IRBP-Exon
1
IRBP-Exon
1
Autosomes
N52
Substitution
(1)a
Substitution
(1)a
N52
Substitution
(1)a
Deletion
(1)a
SINE (1)a
None
N53
None
None
None
Substitution
(1)a
Deletion
(1)a
None
None
None
N50
None
None
None
None
N50
None
Substitution
(2)a
N52
None
None
None
N50
None
None
None
N50
None
Substitution
(1)a
N51
Substitution
(2)a
Substitution
(2)a
Substitution
(1)a
N55
None
None
Substitution
(1)a
Deletion
(2)a
Substitution
(1)a
None
N54
None
None
N50
None
None
None
N50
None
Substitution
(1)a
Substitution
(1)a
None
N52
None
None
Substitution
(6)a
Deletion
(1)a
None
Substitution
(4)a
Deletion
(1)a
None
N50
None
None
None
N50
None
N58
Substitution
(2)a
Substitution
(6)a
N 5 18
None
Substitution
(1)a
NOTE.—N, number of sites (see supplementary table 5 and figs. 1–7 of alignments files, Supplementary Material online).
a
Parentheses are the number of sites showing substitutions, deletions, or SINE insertions compared with other lineage.
Substitution
(3)a
Substitution
(2)a
Deletion
(2)a
Substitution
(1)a
Substitution
(3)a
Deletion
(1)a
Substitution
(2)a
Deletion
(1)a
N51
N54
N55
Sex Chromosome Evolution in Bears 1349
Subfamily Ursinae
ZFY-Final
Intron
X Chromosome Genes
1350 Nakagome et al.
A
Species
86/64/75
0.975
93/98/96
1.00
Ursus arctos
Brown bear
Ursus maritimus
Polar bear
67/-/-
Ursus americanus
94/-/100/100/100
1.00
Ursus ursinus
Ursus malayanus
Ursus thibetanus
American black bear
Sloth bear
Sun bear
Asiatic black bear
Tremarctos ornatus
Spectacled bear
Ailuropoda melanoleuca
Giant panda
0.005
B
Species
72/61/60
1.00
85/87/83
1.00
Ursus arctos
Ursus maritimus
Ursus americanus
99/98/98
1.00
Ursus thibetanus
Brown bear
Polar bear
American black bear
Asiatic black bear
70/-/-
Ursus ursinus
77/88/83
1.00
Ursus malayanus
Tremarctos ornatus
Ailuropoda melanoleuca
Sloth bear
Sun bear
Spectacled bear
Giant panda
0.002
FIG. 1.—Phylogenetic trees for combined data from ZFY-SMCY-SRY (3,238 bp) of Y chromosome genes (A) and ZFX-SMCX-PLP-ALAS2 (4,083
bp) of X chromosome genes (B) for 8 species of Ursidae. Nearly identical topologies were obtained among ME, MP, ML, and Bayesian analysis.
Shown as representative are the ME trees using Tajima–Nei distance model. Under ME, phylogenetic analysis resulted in a single tree for Y
chromosome or X chromosome genes, respectively (tree score was 0.05437 for Y chromosome genes or 0.02948 for X chromosome genes).
Phylogenetic analysis using MP criteria recovered 2 equivalent trees in Y chromosome genes and 1 equivalent tree in X chromosome genes. For ML, ln
likelihood scores were 5327.98091 in Y chromosome genes and 6430.86294 in X chromosome genes. Above numbers in italics adjacent to nodes
represent bootstrap values as ME (1,000 iterations)/MP (1,000 iterations)/ML (100 iterations), and below the numbers show posterior probability
values. A solid asterisk indicates a SINE insertion of SMCY-intron 4 in a common ancestor to Ursus arctos and Ursus maritimus.
and the lowest for those genes located on the X chromosome
(node A to T. ornatus, and each ursine species in table 4
and supplementary fig. 8, Supplementary Material online). These relative differences in substitution rates are not
consistent across the bear phylogeny (table 4 and fig. 4).
In particular, estimates of the relative differences between
Y-linked, X-linked, and autosome substitution rates from
node A to node B were discordant with those based on
node B to each ursine species (table 4 and fig. 4). In the
former, Y-linked genes had significantly higher values
compared with those from the X-linked and autosome
genes (P , 0.01), which themselves evolved at the roughly
same rate. In contrast, substitution rates computed from
node B to each ursine species were not significantly elevated, but rather were appreciably lower in Y-linked genes
relative to autosome genes, and roughly equivalent to the
estimates from X-linked gene regions (table 4 and fig. 4).
Discussion
Patterns of evolution for genes located on the sex chromosomes are assessed within the bear family Ursidae. The
male-determining gene SRY is sequenced in entirety along
with intron segments from X–Y homologs ZFX/ZFY and
SMCX/SMCY and X-linked genes PLP and ALAS2. Phylogenetic analyses of the sex-linked genes indicate low levels
of homoplasy, consistent with previous studies of speciation in the cat family Felidae (Pecon-Slattery and O’Brien
1998; Pecon-Slattery et al. 2004; Johnson et al. 2006; King
Sex Chromosome Evolution in Bears 1351
Species
69/74/78
1.00
Ursus arctos
95/90/87
1.00
Ursus maritimus
Ursus americanus
90/69/73
0.98
53/55/57
0.90
Brown bear
Polar bear
American black bear
Ursus thibetanus
Asiatic black bear
Ursus malayanus
Sun bear
97/90/88
1.00
Ursus ursinus
Sloth bear
Tremarctos ornatus
Spectacled bear
Ailuropoda melanoleuca
Giant panda
0.001
FIG. 2.—The ME trees reconstructed using previously published autosome concatenated sequences IRBP-Exon 1 and TTR-Intron 1 (2,281 bp) of 8
Ursidae species. Genetic distances were calculated using Tajima–Nei distance model. Nearly identical topologies were obtained from MP, ML, and
Bayesian analysis. Shown as representative is the ME tree using Tajima–Nei distance model. Under ME, phylogenetic analysis resulted in a single tree
(tree score was 0.04203). Phylogenetic analysis using MP criteria recovered 1 equivalent tree. For ML, ln likelihood score was 3804.52089. Numbers
in italics above internal branches show the bootstrap values for ME (1,000 iterations)/MP (1,000 iterations)/ML (100 iterations), and below numbers are
posterior probability values by the Bayesian analysis.
et al. 2007), but exhibit unusual patterns of substitution
within the evolution of the bear family.
Phylogenetic Assessment of Y Chromosome and X
Chromosome Genes
Both Y-linked and X-linked genes affirm the early divergence of the spectacled bear (T. ornatus) and the monophyletic lineage of the 6 species of the subfamily Ursinae
observed with phylogenetic analyses of complete mtDNA
(Yu et al. 2007) and autosome genes (Yu et al. 2004). Further, ZFY and SMCY contain SINE elements not found in
their respective X homologs that are phylogenetically informative in bear speciation. First described in a subset of bear
species (Slattery et al. 2000), the present study reveals that
the SINE insertion in the ZFY-final intron occurs in all bear
species and the SINE in SMCY offers further confirmation
of the close association between the polar bear (U. maritimus) and brown bear (U. arctos). Thus, the ZFY SINE is
ancestral to all bears and inserted into the Y chromosome
prior to the divergence of the 8 extant species at least 12
Species
92/92/95
1.00
100/99/100
1.00
81/52/57
0.91
100/100/100
1.00
B
A
97/89/89
1.00
Ursus arctos
Ursus maritimus
Ursus americanus
Ursus thibetanus
Brown bear
Polar bear
American black bear
Asiatic black bear
Ursus malayanus
Sun bear
Ursus ursinus
Sloth bear
Tremarctos ornatus
Spectacled bear
Ailuropoda melanoleuca
Giant panda
0.002
FIG. 3.—Phylogenetic tree of combined nuclear genes data (9,511 bp) from Y chromosome genes (ZFY-SMCY-SRY), X chromosome genes (ZFXSMCX-PLP-ALAS2), and autosome genes (IRBP-TTR) for 8 species of Ursidae. Nearly identical topologies were obtained among ME, MP, ML, and
Bayesian analysis. Shown as representative is the ME tree using Tajima–Nei distance model. Under ME, phylogenetic analysis resulted in a single tree
(tree score was 0.04081). Phylogenetic analysis using MP criteria recovered 2 equivalent trees. The MP tree has a consistency index of 0.962. For ML,
ln likelihood score was 15721.31571. Above numbers in italics adjacent to nodes represent bootstrap values as ME (1,000 iterations)/MP (1,000
iterations)/ML (100 iterations), and below the numbers show posterior probability values. Node A shows the ancestral node of Ursidae excluding the
giant panda, and node B indicates the ancestral node of the subfamily Ursinae.
0.473
0.368
0.422
0.302
0.326
0.482
0.722
0.687
0.707
0.535
0.627
0.917
0.277
0.118
0.198
0.118
0.091
0.144
0.194
0.137
0.203
0.042
0.211
0.177
0.175
0.116
0.057
0.001c
0.293
0.058
0.350
0.112
0.221
0.024
0.219
0.288
0.186
0.186
0.498
0.002c
0.169
0.366
0.059
0.178
0.297
0.178
0.058
0.176
c
Concatenating 4 (Y chromosome), 4 (X chromosome), and 2 (autosomes) genes.
The average substitution rates from node to each Ursine species.
The negative values of branch lengths.
b
0.149
0.005c
0.148
0.14
0.305
0.304
0.236
0.236
0.480
0.008c
0.357
0.358
0.290
0.290
0.551
0.091
0.495
0.065
0.339
0.169
0.169
0.001c
0.003c
0.167
0.784
0.577
0.181
0.395
0.763
0.865
0.166
0.699
0.798
0.352
0.195
0.158
0.362
0.324
0.163
0.161
0.233
0.234
0.118
0.116
0.749
0.506
0.304
0.202
0.222
0.456
0.222
0.234
0.256
0.216
0.059
0.158
0.989
0.770
0.534
0.235
0.914
0.633
0.458
0.175
0.905
0.812
0.536
0.277
1.293
0.899
0.602
0.297
0.677
0.647
0.507
0.140
0.255
0.190
0.373
0.059
0.322
0.214
a
Branches
to the spectacled bear
to Ursineb
to B
to Ursineb
to each species
Brown bear
Polar bear
American black bear
Asiatic black bear
Sun bear
Sloth bear
A
A
A
B
B
Autosomesa
TTRIntron 1
ALAS2-Intron 7
PLPIntron 3
X Chromosome Genes
SMCXIntron 4
ZFX-Final
Intron
Y
Chromosomea
SRYCoding
SRYNoncoding
Y Chromosome Genes
SMCYIntron 4
ZFY-Final
Intron
Table 4
Substitution Rates (numbers per 100 sites) of Nuclear Genes (Y chromosome, X chromosome, and autosomes)
X
Chromosomea
IRBPExon 1
Autosome Genes
1352 Nakagome et al.
MYA (Wayne et al. 1991), and the SMCY SINE is more
recent, supporting the view that the polar bear evolved from
brown bear populations isolated in northernmost areas of
Asia during the last glaciations and rapidly adapted to extreme environmental conditions (Kurten 1964; Kurten
1968; Talbot and Shields 1996a). This close association between U. arctos and U. maritimus is clearly supported not
only by patterns of substitution of sex-linked genes examined in this study but also in previous molecular genetic
studies as well (Talbot and Shields 1996a, 1996b; Waits
et al. 1999; Yu et al. 2004, 2007).
Within the subfamily Ursinae, 2 lineages are recovered, the first, well-supported by both Y- and X-linked
genes and comprised of sister taxa of the brown bear (U.
arctos) and polar bear (U. maritimus) with the American
black bear (U. americanus) having a relatively basal position within this clade. The second lineage is less resolved
and is collapsed into a trichotomy of the sun bear (U. malayanus), sloth bear (U. ursinus), and Asiatic black bear (U.
thibetanus). These findings differ from those based on autosome genes that separate Ursinae into 2 clades: one composed of the 2 Asian species and the other formed by the
remaining 4 species (fig. 2). In addition, previously published phylogenetic trees based on mtDNA (Talbot and
Shields 1996a; Waits et al. 1999; Yu et al. 2007) and autosome genes (Yu et al. 2004), as well as morphological
characters (Allen 1938) and the fossil record (Kurten
1964) explicitly define the monophyly for the Asiatic black
bear (U. thibetanus) and American black bear (U. americanus) as sister taxa.
These phylogenetic discrepancies between sex-linked
genes and autosome genes, as well as mtDNA genomes (Yu
et al. 2007), may be due to an insufficient accumulation of
informative substitutions in the subfamily Ursinae. Combined nuclear data (9,515 bp) provides a more resolved
phylogeny (fig. 3) marked by high nodal support and little
homoplasy but still does not recover the expected sister–
taxa relationship of the 2 species of black bear. Rather,
the observed association between the Asiatic black bear
(U. thibetanus) with the sloth bear (U. ursinus) and sun bear
(U. malayanus) is supported mostly by diagnostic changes
within X-linked genes (N 5 2) (fig. 1B, table 3 and supplementary table 5, Supplementary Material online). To investigate the possible influence of incongruent phylogenetic
information of X chromosome, we reconstructed the phylogeny of nuclear genes without the X-linked data (supplementary fig. 9, Supplementary Material online). The
resultant topology is completely consistent with that based
on total combined data indicating that the split of the clade
of 2 black bears is not attributable to incongruent X-linked
partitions but instead reflects the strong association between
the American black bear (U. americanus) with the sister
taxa clade of the brown bear (U. arctos) and polar bear
(U. maritimus) (figs. 1A and B; table 3 and supplementary
table 5, Supplementary Material online). Interestingly, the
identical topology is recovered in a parallel study (Pages et
al. 2007) based on combined data from 14 nuclear genes
including 3 Y-linked genes of ZFY, SRY, and UBE1Y.
Therefore, there is no indication of chromosome bias in lineage definitions and phylogenetic information present
within Y-linked, X-linked, or autosome genes is reflected
Sex Chromosome Evolution in Bears 1353
1
0.8
**
0.6
Y>X, Y>A
*
A>X
0.4
0.2
0
A to B
U. arctos
U. maritimus U. americanus U. thibetanus U. malayanus U. ursinus All ursine bears
Node B to each ursine species
FIG. 4.—Substitution rates of Y chromosome (black), X chromosome (white), and autosome (gray) genes in the branch of node A to B and node B
to each species of ursine (fig. 3) are constructed using the values shown in table 4. Substitution rate values indicate the number of substitutions per 100
sites. In the graphs, the comparisons between chromosomes (Y . X, Y . A, or A . X) above the bars show the significant differences of the
substitution rates (double asterisks are P , 0.01, and single asterisk is P , 0.05) between chromosomes (Y: Y chromosome, X: X chromosome, and
A: autosomes).
equivalently in the nuclear gene tree (fig. 3; table 3 and supplementary table 5, Supplementary Material online).
Differential Rates of Substitution between Y
Chromosome, X Chromosome, and Autosome Genes
Consistent with expectations under the hypothesis of
male-driven evolution (Haldane 1947; Miyata et al. 1987),
the overall average substitution rates of Y chromosome genes
are the highest among nuclear genes (table 4 and supplementary fig. 8, Supplementary Material online) and observed other
mammalian taxa such as primates (Huang et al. 1997; Makova
and Li 2002), carnivores (Pecon-Slattery and O’Brien 1998),
perissodactyls (Goetting-Minesky and Makova 2006), and
rodents (Sandstedt and Tucker 2005). These results also demonstrate that the X-linked genes have the lowest and autosome
genes intermediate rates of substitution (table 4 and supplementary fig. 8, Supplementary Material online). A notable exception to the expected rates of change with Y . X and Y . A
occursinevolutionofthesubfamilyUrsinae(table4andfig.4).
Across the entire bear family, the ratio of Y/X 5 1.933 (95%
confidence interval [CI]: 1.262–2.605) (the equation: V(Y) 5
Y(1 Y)/[L(1 4Y/3)2], V(X) 5 X(1 X)/[L(1 4X/3)]2,
V(Y/X) 5 V(Y)/E(X)2 þ E(Y)2V(X)/E(X)4, and Y/X 5
Y/X 1.96s and Y/Xþ 5 Y/X þ 1.96s in accordance
with Sandstedt and Tucker [2005]) and a 5 3.624 (95%
CI: 1.451–13.176) (the equation: Y/X 5 3 a/(2 þ a) shown
in Miyata et al. [1987]) changes within Ursinae whereby
Y/X 5 1.313 (95% CI: 0.680–1.945) and a 5 1.556 (95%
CI: 0.587–3.687). Thus, possible male-driven evolution is
highly supported in the deeper nodes of the phylogeny but
not in the more recent Ursinae.
Low Divergence of Y Chromosome Genes within
Ursinae
We explore 3 possible explanations for the discordant
evolutionary rates observed with the Y chromosome in bear
evolution (table 4 and fig. 4) namely, 1) biogeographical
effects, 2) evolutionary characteristics of the Y chromosome, and 3) behavioral traits in the natural history of
the subfamily Ursinae. Modern species of Ursinae evolved
recently and rapidly during the Pliocene and Pleistocene
(Yu et al. 2007) and likely experienced dramatic climatic
changes linked with glaciations. For example, the brown
bear (U. arctos) is distributed throughout North America,
Europe, and Asia yet has unique phylogeographic patterns
of extirpation and recolonization linked with patterns of
glaciations (Hofreiter et al. 2004; Miller et al. 2006; Stoen
et al. 2006). Stochastic changes brought about by population expansion, migration, and contraction during this time
may have facilitated selective sweeps in the Y chromosome
that led to less than expected substitution rates in Ursinae
observed today. The effective population size of Y chromosome is smaller than autosomes (Lawson Handley and
Perrin 2007). Therefore, if a favored mutation occurred
on the Y chromosome in ancestral populations undergoing
frequent structural changes, then the allele might spread
more rapidly than expected. The replacement of a circulating Y chromosome with another within ancestral populations of Ursinae would result in the appearance of low
levels of divergence between species observed here.
In contrast to the fixation of favorable mutations resulting in selective sweeps within a species, reduced genetic
diversity on the Y chromosome is influenced also by the
lack of conventional recombination during meiosis. A
1354 Nakagome et al.
positive correlation exists between recombination rate and
polymorphism within genomes (Innan and Stephan 2003).
Consequently, due to the lack of recombination needed to
remove harmful mutations, the fate of genes on the Y chromosome would depend on the inexorable accumulation
of mutations (of which some will be deleterious), due to
Muller’s ratchet (Charlesworth B and Charlesworth D
1997). Advantageous mutations on the Y chromosome
could cause the fixation of all deleterious mutations present
on the chromosome, and successive ‘‘selective sweeps’’ of
this kind would cause the fixation of deleterious alleles
at many Y-linked loci (genetic hitchhiking). Background
or purifying selection acting against these harmful mutations could reduce the number of Y chromosome variants
within a species, leading to decreased diversity of the Y
chromosome in a finite population.
As described above, the biogeographical effects experienced by ursine species combined with the nonrecombining
characteristics of the Y chromosome could be the basis for
low divergence of Y-linked genes in Ursinae. However, if
these 2 factors alone regulate Y chromosome divergence,
then other mammalian species within these same biogeographical regions should show similar patterns because these
processes are not specific to bears. For example, the divergence of the 7 species within the domestic cat lineage and the
5 species of the leopard cat lineage of Felidae occurred
roughly at the same time as Ursinae and within similar biogeographic zones (Johnson et al. 2006). However, these felid
lineages do not show reduced diversity on the Y chromosome but instead are defined by unique diagnostic substitutions (Pecon-Slattery et al. 2004) correlated with increased
rates of substitution relative to genes located on autosomes
and the X chromosome (Johnson et al. 2006).
We propose that social behavior may be a significant
factor in the discordant patterns of genome evolution in
bears. The Ursinae, like most large mammals, exhibit
male-biased dispersal and female philopatry (Matsuhashi
et al. 1999; Nowak 1999; McLellan and Hovey 2001;
Kojola et al. 2003; Onorato et al. 2004; Stoen et al.
2005, 2006). Migration caused by male-biased dispersal allows gene flow and is a potent force in homogenizing genetic divergence among subpopulations. In addition, the
reproductive system of bears is polygynandrous by which
a female may mate with 2 or more males, who themselves
may pair with several different females (Nowak 1991).
Therefore, if the social structure of female philopatry,
male-biased dispersal, and polygynandry was present in ancestral populations of Ursinae, relatively low number of
male breeders and high male migration rates would result
in low effective population size and reduction of genetic
diversity for the Y chromosome among breeding groups
(Chesser and Baker 1996). Furthermore, female philopatry
has been shown to increase the effective population size of
mtDNA by one-half relative to autosomes and almost 6
times relative to the Y chromosome (Chesser and Baker
1996). Thus, genetic diversity of the Y chromosome among
ancestral populations would be lost at a faster rate than
mtDNA and autosomes. These results were also supported
in Laporte and Charlesworth (2002) showing that under
predominantly male migration, high genetic differentiation
was obtained in mtDNA, followed by Y chromosome, X
chromosome, and autosomes. In addition, Hoelzer (1997)
and Hoelzer et al. (1998) showed that nodes within mitochondrial gene trees were deeper than those in nuclear gene
trees if the female migration rate was low. Lastly, the effect
of behavioral traits on the effective population size of autosomes may not be associated with differences in dispersal
between sexes but rather male polygyny (Chesser and
Baker 1996). Therefore, we suggest low divergence of
Y-linked genes observed here compared with high divergence of mtDNA for the subfamily Ursinae (Yu et al.
2007) may be due, in part, to male migration, female philopatry, and polygynandry during ursine bear speciation.
Conclusions
This work constitutes the first direct comparisons of Y
chromosome, X chromosome, autosomes, and mtDNA
phylogenies from the bear family Ursidae for the purpose
of evaluating the influence of social structure into species
divergence. Although the Y chromosome has the highest
substitution rates in the deeper nodes of the bear phylogeny,
low rates are shown in evolution of the subfamily Ursinae.
We suggest 3 possibilities for these results: biogeographical
effects such as iterative glaciations of the recent past, molecular evolutionary characters of the Y chromosome, and
behavioral traits of sex-biased dispersal and reproductive
system. Although all these explanations combined are significant, we propose that dispersal patterns and mating systems should be strongly considered when reconstructing
molecular evolutionary history of mammals, especially in
those with the matriarchal social structures.
Supplementary Material
Supplementary figures 1–9 and tables 1–5 are available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).
Acknowledgments
We would like to thank Dr T. Mano (Hokkaido Institute of Environmental Science), K. Ito and M. Kasahara
(Ueno Zoological Gardens), J. Morita (Ikeda Zoo), Dr K.
Murata (Nihon University), K. Takami (Tennoji Zoo),
M. Ueda (Yokohama Zoo), and S. Dakemoto for supplying
bear samples. We also appreciate sample preparations by
C. Nishida (Hokkaido University). Our thanks go to Dr S. A.
Sandstedt (Michigan University), Dr Y. Ishibashi (Forest
and Forest Products Research Institute), and Dr H. Tsuruga
(Hokkaido Institute of Environmental Science) for helpful
suggestions. This study was supported by Grants-in-Aid for
Scientific Research from the Japan Society for the Promotion of Science and by the 21st Century Center of Excellence Program ‘‘Neo-Science of Natural History’’ at
Hokkaido University financed from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Literature Cited
Allen GM. 1938. The mammals of China and Mongolia. New
York: American Museum of Natural History.
Sex Chromosome Evolution in Bears 1355
Charlesworth B. 1991. The evolution of sex chromosomes.
Science. 251:1030–1033.
Charlesworth B. 1996. The evolution of chromosomal sex
determination and dosage compensation. Curr Biol. 6:149–162.
Charlesworth B, Charlesworth D. 1997. Rapid fixation of
deleterious alleles can be caused by Muller’s ratchet. Genet
Res. 70:63–73.
Chesser RK, Baker RJ. 1996. Effective sizes and dynamics of
uniparentally and diparentally inherited genes. Genetics.
144:1225–1235.
Eriksson J, Siedel H, Lukas D, Kayser M, Erler A, Hashimoto C,
Hohmann G, Boesch C, Vigilant L. 2006. Y-chromosome
analysis confirms highly sex-biased dispersal and suggests
a low male effective population size in bonobos (Pan
paniscus). Mol Ecol. 15:939–949.
Goetting-Minesky MP, Makova KD. 2006. Mammalian male
mutation bias: impacts of generation time and regional
variation in substitution rates. J Mol Evol. 63:537–544.
Haldane JBS. 1947. The mutation rate of the gene for
haemophilia and its segregation ratios in males and females.
Ann Eugen. 13:158–163.
Hammond RL, Handley LJ, Winney BJ, Bruford MW, Perrin N.
2006. Genetic evidence for female-biased dispersal and gene
flow in a polygynous primate. Proc Biol Sci. 273:479–484.
Hoelzer GA. 1997. Inferring phylogenies from mtDNA variation:
mitochondrial-gene trees versus nuclear-gene trees revisited.
Evolution. 51:622–626.
Hoelzer GA, Wallman J, Melnick DJ. 1998. The effects of social
structure, geographical structure, and population size on the
evolution of mitochondrial DNA: II. Molecular clocks and the
lineage sorting period. J Mol Evol. 47:21–31.
Hofreiter M, Serre D, Rohland N, Rabeder G, Nagel D,
Conard N, Munzel S, Paabo S. 2004. Lack of phylogeography
in European mammals before the last glaciation. Proc Natl
Acad Sci USA. 101:12963–12968.
Huang W, Chang BH, Gu X, Hewett-Emmett D, Li W. 1997. Sex
differences in mutation rate in higher primates estimated from
AMG intron sequences. J Mol Evol. 44:463–465.
Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian inference
of phylogenetic trees. Bioinformatics. 17:754–755.
Innan H, Stephan W. 2003. Distinguishing the hitchhiking and
background selection models. Genetics. 165:2307–2312.
Johnson WE, Eizirik E, Pecon-Slattery J, Murphy WJ,
Antunes A, Teeling E, O’Brien SJ. 2006. The late Miocene
radiation of modern Felidae: a genetic assessment. Science.
311:73–77.
King V, Goodfellow PN, Pearks Wilkerson AJ, Johnson WE,
O’Brien SJ, Pecon-Slattery J. 2007. Evolution of the maledetermining gene SRY within the cat family Felidae.
Genetics. 175:1855–1867.
Kojola I, Danilov PI, Hanna-Mari L, Vladimir B, Andrei Y.
2003. Brown bear population structure in core and periphery:
an analysis of human statistics from Russian Karelia and
Finland. Ursus. 14:17–20.
Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software
for molecular evolutionary genetics analysis and sequence
alignment. Brief Bioinform. 5:150–163.
Kurten B. 1964. The evolution of the polar bear, Ursus maritimus
Phipps. Acta Zool Fenn. 108:1–30.
Kurten B. 1968. Pleistocene mammals of Europe. Chicago (IL):
Aldine.
Laporte V, Charlesworth B. 2002. Effective population size and
population subdivision in demographically structured populations. Genetics. 162:501–519.
Lawson Handley LJ, Perrin N. 2007. Advances in our understanding of mammalian sex-biased dispersal. Mol Ecol.
16:1559–1578.
Makova KD, Li WH. 2002. Strong male-driven evolution of
DNA sequences in humans and apes. Nature. 416:624–626.
Matsuhashi T, Masuda R, Mano T, Yoshida MC. 1999.
Microevolution of the mitochondrial DNA control region in
the Japanese brown bear (Ursus arctos) population. Mol Biol
Evol. 16:676–684.
Mazza P, Rustioni M. 1994. On the phylogeny of Eurasian bears.
Palaeontographica Abt A. 230:1–38.
McLellan BN, Hovey FW. 2001. Natal dispersal of grizzly bears.
Can J Zool. 79:838–844.
Miller CR, Waits LP, Joyce P. 2006. Phylogeography and
mitochondrial diversity of extirpated brown bear (Ursus
arctos) populations in the contiguous United States and
Mexico. Mol Ecol. 15:4477–4485.
Miyata T, Hayashida H, Kuma K, Mitsuyasu K, Yasunaga T.
1987. Male-driven molecular evolution: a model and
nucleotide sequence analysis. Cold Spring Harb Symp Quant
Biol. 52:863–867.
Nash WG, O’Brien SJ. 1987. A comparative chromosome
banding analysis of the Ursidae and their relationship to other
carnivores. Cytogenet Cell Genet. 45:206–212.
Nash WG, Wienberg J, Ferguson-Smith MA, Menninger JC,
O’Brien SJ. 1998. Comparative genomics: tracking chromosome evolution in the family Ursidae using reciprocal
chromosome painting. Cytogenet Cell Genet. 83:182–192.
Nowak RM. 1991. Walker’s mammals of the world, 5th ed.
Baltimore (MD): Johns Hopkins Press.
Nowak RM. 1999. Walker’s mammals of the world, 6th ed.
Baltimore (MD): Johns Hopkins.
Onorato DP, Hellgren EC, Bussche RAVD, Raymond Skiles JJ.
2004. Paternity and relatedness of American black bears
recolonizing a desert montane island. Can J Zool.
82:1201–1210.
Oota H, Settheetham-Ishida W, Tiwawech D, Ishida T,
Stoneking M. 2001. Human mtDNA and Y-chromosome
variation is correlated with matrilocal versus patrilocal
residence. Nat Genet. 29:20–21.
Pages M, Sebastien C, Catherine K, Mathilde P, Sandrine H,
Catherine H. 2007. Combined analysis of fourteen nuclear
genes refines the Ursidae phylogeny. Mol Phylogenet Evol.
doi: 10.1016/j.ympev.2007.10.019.
Pecon-Slattery J, O’Brien SJ. 1998. Patterns of Y and X
chromosome DNA sequence divergence during the Felidae
radiation. Genetics. 148:1245–1255.
Pecon-Slattery J, Pearks Wilkerson AJ, Murphy WJ, O’Brien SJ.
2004. Phylogenetic assessment of introns and SINEs within
the Y chromosome using the cat family Felidae as a species
tree. Mol Biol Evol. 21:2299–2309.
Posada D, Crandall KA. 1998. MODELTEST: testing the model
of DNA substitution. Bioinformatics. 14:817–818.
Rappold GA. 1993. The pseudoautosomal regions of the human
sex chromosomes. Hum Genet. 92:315–324.
Sandstedt SA, Tucker PK. 2005. Male-driven evolution in
closely related species of the mouse genus Mus. J Mol Evol.
61:138–144.
Seielstad MT, Minch E, Cavalli-Sforza LL. 1998. Genetic
evidence for a higher female migration rate in humans. Nat
Genet. 20:278–280.
Shimmin LC, Chang BH, Li WH. 1993. Male-driven evolution of
DNA sequences. Nature. 362:745–747.
Slattery JP, Murphy WJ, O’Brien SJ. 2000. Patterns of diversity
among SINE elements isolated from three Y-chromosome
genes in carnivores. Mol Biol Evol. 17:825–829.
Slattery JP, O’Brien SJ. 1995. Molecular phylogeny of the red
panda (Ailurus fulgens). J Hered. 86:413–422.
Snedecor GW, Cochran WG. 1989. Statistical methods. Ames
(IA): Iowa State University Press.
1356 Nakagome et al.
Stoen O-G, Bellemain E, Saebo S, Swenson JE. 2005. Kinrelated spatial structure in brown bears Ursus arctos. Behav
Ecol Sociobiol. 59:191–197.
Stoen OG, Zedrosser A, Saebo S, Swenson JE. 2006. Inversely
density-dependent natal dispersal in brown bears Ursus
arctos. Oecologia. 148:356–364.
Swofford DL. 2002. PAUP*: phylogenetic analysis using
parsimony (* and other methods). Version 4.0b10. Sunderland
(MA): Sinauer.
Tajima F, Nei M. 1984. Estimation of evolutionary distance
between nucleotide sequences. Mol Biol Evol. 1:269–285.
Talbot SL, Shields GF. 1996a. A phylogeny of the bears
(Ursidae) inferred from complete sequences of three mitochondrial genes. Mol Phylogenet Evol. 5:567–575.
Talbot SL, Shields GF. 1996b. Phylogeography of brown bears
(Ursus arctos) of Alaska and paraphyly within the Ursidae.
Mol Phylogenet Evol. 5:477–494.
Waits LP, Sullivan J, O’Brien SJ, Ward RH. 1999. Rapid
radiation events in the family Ursidae indicated by likelihood
phylogenetic estimation from multiple fragments of mtDNA.
Mol Phylogenet Evol. 13:82–92.
Wayne RK, Van Valkenburgh B, O’Brien SJ. 1991. Molecular
distance and divergence time in carnivores and primates. Mol
Biol Evol. 8:297–319.
Wolfe KH, Li WH. 2003. Molecular evolution meets the
genomics revolution. Nat Genet. 33(Suppl):255–265.
Yu L, Li QW, Ryder OA, Zhang YP. 2004. Phylogeny of the
bears (Ursidae) based on nuclear and mitochondrial genes.
Mol Phylogenet Evol. 32:480–494.
Yu L, Li YW, Ryder OA, Zhang YP. 2007. Analysis of complete
mitochondrial genome sequences increases phylogenetic
resolution of bears (Ursidae), a mammalian family that
experienced rapid speciation. BMC Evol Biol. 7:198.
John H McDonald, Associate Editor
Accepted March 26, 2008