MBE Advance Access originally published online on April 9, 2008
Molecular Biology and Evolution 2008 25(7):1344-1356; doi:10.1093/molbev/msn086
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Research Articles |
Unequal Rates of Y Chromosome Gene Divergence during Speciation of the Family Ursidae
* Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Japan
Laboratory of Genomic Diversity, National Cancer Institute-Frederick, Frederick, MD
Department of Genome Dynamics, Creative Research Initiative "Sousei", Hokkaido University, Sapporo, Japan
E-mail: masudary{at}ees.hokudai.ac.jp.
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Abstract |
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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.
Key Words: Ursidae • sex-linked genes • male-biased dispersal • female philopatry • matriarchal structure
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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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 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 X-linked (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).
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 male-biased dispersal.
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Materials and Methods |
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DNA Specimens
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.
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Primer Design and Polymerase Chain Reaction Analysis of 3 Y Chromosome Genes and 4 X Chromosome Genes
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 (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 [GenBank] /NM_011768 in the mouse; SMCY/X, NM_004653 [GenBank] /NM_004187 in the human and NM_011419 [GenBank] /NM_0137668 in the mouse; PLP, NM_199478 [GenBank] .1 in the human and NM_011123 [GenBank] .2 in the mouse; and ALAS2, NM_000032 [GenBank] .2 in the human and NM_009653 [GenBank] .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 µl including 2 µl of the DNA extract (50–100 ng/µl), 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphate, and 0.25 µM 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 U-SMC-2F/2R). For ZFY/X, it was necessary to perform nested PCR using 5 µl 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 [GenBank] ) were used to design 2 primer pairs for seminested PCR; U-SRY-1F/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 = 0.3004, C = 0.1730, G = 0.2044, and T = 0.3222; a transition:transversion ratio = 1.7417 for Y-linked genes; 2) the HKY + gamma model with estimated nucleotide base frequencies of A = 0.3054, C = 0.1919, G = 0.2239, and T = 0.2788; a transition:transversion ratio = 2.1345; gamma = 0.0139 for X-linked genes; 3) the HKY + gamma model with estimated nucleotide base frequencies of A = 0.2208, C = 0.2867, G = 0.2692, and T = 0.2233; a transition:transversion ratio = 3.021; gamma = 0.006 for autosome genes; 4) HKY + gamma model with estimated nucleotide base frequencies of A = 0.2836, C = 0.2084, G = 0.2281, and T = 0.2800; a transition:transversion ratio = 2.1748; gamma = 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 of derived phylogenetic relationships was evaluated using bootstrap analyses, and clades supported by greater than 50% of node bootstrap values were retained. For the ME and MP analyses, 1,000 iterations of bootstrap were performed with heuristic 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, 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 Y-linked, 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 least-significant difference [PLSD]). Significance levels of P < 0.05 and P < 0.01 were adopted throughout.
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Results |
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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 ALAS2-intron 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 occurred at nt 2,337–2,398 in the ALAS2-intron 7 (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 = 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).
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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 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.
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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).
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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 = 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 = 2 in the former lineage and N = 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 in Y-linked genes and N = 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 = 2) and autosome genes (N = 1) or only autosome genes (N = 1). The monophyly of U. arctos and U. maritimus was supported consistently across all nuclear genes within Ursinae (N = 3 in Y-linked, N = 2 in X-linked, and N = 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 = 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, 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).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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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 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 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 = 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 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 occurs in evolution of the subfamily Ursinae (table 4 and fig. 4). Across the entire bear family, the ratio of Y/X = 1.933 (95% confidence interval [CI]: 1.262–2.605) (the equation: V(Y) = Y(1 – Y)/[L(1 – 4Y/3)2], V(X) = X(1 – X)/[L(1 – 4X/3)]2, V(Y/X) = V(Y)/E(X)2 + E(Y)2V(X)/E(X)4, and Y/X– = Y/X – 1.96s and Y/X+ = Y/X + 1.96s in accordance with Sandstedt and Tucker [2005]) and 
= 3.624 (95% CI: 1.451–13.176) (the equation: Y/X = 3 /(2 + ) shown in Miyata et al. [1987]) changes within Ursinae whereby Y/X = 1.313 (95% CI: 0.680–1.945) and = 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 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.
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Conclusions |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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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.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures 1–9 and tables 1–5 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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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.
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1 Present address: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan.
John H McDonald, Associate Editor
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