BACKGROUND
understanding how levels of genetic variation influence the survival of threatened species is of fundamental interest to evolutionary and conservation biologists because many natural populations are threatened by intense reduction and fragmentation of habitat, leading to isolation, declining populations, and decreasing genetic diversity  <cit> .
loss of genetic diversity may increase the risk of extinction due to decreased reproductive fitness, decreased adaptive flexibility, and increased disease susceptibility  <cit> .
clarifying the mechanism that determines genetic variation in small, isolated populations is therefore essential for their conservation  <cit> .
an important assumption in conservation genetics is that small, isolated populations are more sensitive to genetic drift and inbreeding  <cit> .
genetic drift is the random fluctuation of allele frequencies over time; thus, adaptive alleles may be lost and deleterious alleles could be fixed in the population.
the small population size and fixation of deleterious alleles leads to inbreeding depression and reduction of individual fitness, which decreases viability and compromises a population’s evolutionary adaptive potential  <cit> .
however, some functionally important genes that are maintained by balancing selection, such as major histocompatibility complex  genes, may have a different evolutionary pattern compared with neutral markers.
the multi-gene mhc family is found in vertebrates, codes for cell surface glycoproteins, and is important in animal conservation due to its role in resisting pathogens  <cit> .
compared to nearly neutral markers such as microsatellite loci or mitochondrial dna, which are informative for phylogenetic and phylogeographic reconstructions  <cit> , mhc variability is believed to determine the capability of individuals to resist continuously evolving pathogens and parasites.
consequently, mhc variability is a reflection of the processes that are related to adaptive evolution within and between populations  <cit> .
thus, most variation at mhc loci reflects the effects of balancing selection  <cit> , which is the main mechanism for retaining high mhc genetic diversity.
balancing selection includes frequency-dependent selection, overdominance and diversifying selection and promotes long evolutionary persistence of individual alleles and strongly differentiated allelic lineages in mammals  <cit> .
besides balancing selection, intragenic recombination has also been suggested as one evolutionary mechanism for generation of mhc sequence diversity  <cit> .
still, several significant questions remain.
current results conflict or are unclear with regard to the relative roles of balancing selection and genetic drift in maintaining mhc polymorphism in small, isolated, or severely bottlenecked populations  <cit> .
studies of guppies, mexican wolves and namibian leopards found that even when genetic diversity at neutral markers was poor, polymorphisms in mhc were still maintained by balancing selection  <cit> .
in contrast, other research has found low levels of detectable polymorphisms for mhc genes in populations with lower diversity in neutral markers, including studies on fallow deer   <cit> , northern elephant seals   <cit> , great crested newt   <cit> , and the black-footed rock-wallaby   <cit> .
these results suggest that compared to genetic drift balancing selection is relatively weak in small populations, leading to reduced variation at some mhc loci  <cit> .
to elucidate the role of balancing selection and genetic drift in populations, fst outlier analysis  <cit>  is widely used because demographic processes affect neutral loci and lead to population differentiation   <cit> .
the fst values are computed for all genes to distinguish the genes under selection from those non-selective genes  <cit> .
we studied mhc and neutral genetic variation in the golden snub-nosed monkey , an endangered primate endemic to china where it inhabits three isolated areas: sichuan and gansu provinces ; the qinling mountains, shaanxi province  and the shennongjia forestry district, hubei province  .
current census data suggest that fewer than  <dig>  individuals remain   <cit> , and mitochondrial dna and microsatellite studies indicate that the genetically distinct snj population has very low genetic variation  <cit> .
the current effective population size  in snj is approximately  <dig> individuals  <cit> .
the small size and isolation that typically characterize local populations make the golden snub-nosed monkey particularly susceptible to loss of genetic variation through inbreeding and genetic drift  <cit> .
in this study, we 1) investigated population structure and polymorphism levels of  <dig> microsatellite loci and  <dig> mhc ii genes, 2) tested for both selection at the mhc loci and patterns of between population differentiations, and 3) evaluated the role of balancing selection and genetic drift in populations.
RESULTS
genetic variation at microsatellites
genotyping of the  <dig> microsatellite loci revealed that the highest level of expected heterozygosity  and observed heterozygosity  was in the sg population, while the lowest level was in the snj population .
the hardy–weinberg equilibrium test showed no deviation from equilibrium after bonferroni correction.
no linkage disequilibrium occurred for pairs of loci in any population, and micro-checker revealed no evidence of stuttering, null alleles, and allele dropout.
ar, allelic richness; he, expected heterozygosity; ho, observed heterozygosity; fis, inbreeding coefficient.
mhc variation, expression analyses, and recombination analysis
for each locus, we analysed  <dig> clones from  <dig> individuals that represented three monkey populations.
we obtained  <dig> different dqa <dig> fragments with lengths that ranged from  <dig> bp to  <dig> bp including exon  <dig> and partial intron  <dig> and intron  <dig> .
after performing blast with the hla-dqa <dig> and macaca mulatta dqa <dig> exon  <dig>  we removed the intron and obtained  <dig> dqa <dig> exon  <dig> sequences with an equal length of  <dig> bp.
these sequences were labelled as rhro-dqa1*01– <dig>  according to the nomenclature of klein et al.
<cit>  and were used for analysis.
at the dqb <dig> locus, we identified  <dig> unique dqb <dig> exon  <dig> sequences that we called rhro-dqb1*01– <dig> .
fragments of mhc loci were successfully amplified from the cdna of  <dig> blood samples that were also used in dna amplifications.
at the dqa <dig> locus,  <dig> bp fragments of dqa <dig> exon  <dig> were obtained from  <dig> blood samples, and these fragments were part of the alleles obtained from dna in the same individual .
at the dqb <dig> locus longer fragments were obtained, including the entire exon  <dig>  partial exon  <dig>  and exon 3; however, these fragments were only obtained from  <dig> blood sample .
shorter fragments of dqb <dig> exon  <dig> were obtained from  <dig> blood samples .
no sequences contained stop codons.
the sequences from rna were identical with that from dna, which supports the hypothesis that these mhc pcr products were transcribed and expressed.
no more than two alleles were present per individual, suggesting that for each mhc locus one gene copy was sequenced with these primer sets.
no stop codon or insertion/deletion was detected in dqa <dig> and dqb <dig>  the measures of mhc diversity in the  <dig> populations are summarized in table  <dig>  all three populations shared four of the nine dqa <dig> alleles and two of the among  <dig> dqb <dig> alleles .
the mhc sequences were highly divergent:  <dig> of  <dig> bp of dqa <dig> and  <dig> of  <dig> bp of dqb <dig> were variable.
the hardy–weinberg equilibrium tests showed that the ql population deviated from equilibrium .
linkage disequilibrium occurred in the sg population .
population recombination analysis using ldhat revealed that the dqb <dig> locus had a higher recombination rate  when all alleles were included between the mhc loci .
private alleles, alleles only found in this population; h, genetic variation; ar, allele richness; fis, inbreeding coefficient.
population structure and phylogenetic analysis
microsatellite-based analyses detected significant genetic structures among the populations .
two genetic clusters were found  with a clear maximum Δk .
the pairwise fst was the largest between snj and the other  <dig> populations among all of the fst values , which showed that the differences between sg and ql were minimal compared to those between sg-ql and snj.
however, there was also a small Δk peak at k =  <dig> , and results showed that in addition to the divergence between sg and ql, there was also divergence within the sg and ql populations.
these results agree with earlier studies, where the differences within the sg and ql populations resulted from their different origins and habitat fragmentation  <cit> .
the clear population structure observed in microsatellites was not found at either the dqa <dig> locus or the dqb <dig> locus .
similar to previous studies  <cit> , the relationship among mhc alleles did not agree with the geographical locations of the  <dig> populations, as observed in the microsatellite results .
dqa1/dqb <dig> alleles were not grouped by species but were mixed with other species .
no clear clade could be identified from the phylogenetic tree at these two loci.
populations had more private alleles at dqb <dig> than dqa <dig> .
four among  <dig> dqa <dig> exon  <dig> alleles were commonly shared among the  <dig> populations, while just  <dig>  among  <dig> dqb <dig> exon  <dig> alleles were shared among all of the populations .
however, frequency differences of these shared alleles were found between all population pairs .
comparisons of pairwise fst values of mhc and microsatellite sites are shown in figure  <dig>  the fst values showed lower divergence among populations compared with microsatellite loci .
half of the fst values at the mhc loci were higher than  <dig> , indicating that differences existed among populations, especially between sg and snj.
the population difference was more distinct at the dqb <dig> locus: the differentiations between the sg and snj populations and between the ql and snj populations were even larger than the differentiation at the microsatellite.
historical selection
evidence for historical balancing selection was detected.
first, in the abs, the number of nonsynonymous  mutations was greater than the number of synonymous  mutations using the z-test  .
although p-value was not significant for dqa <dig> locus in mega, abs sites under significant selection were detected in paml  <dig> .
after comparisons of various codon evolution models using codeml in paml  <dig>  models integrating positive selection  matched mhc better than the other models based on the akaike information criterion  values .
under model m2a,  <dig> dqa <dig> sites and  <dig> dqb <dig> sites were exposed to significant selection.
under model m <dig>   <dig> dqa <dig> sites and  <dig> dqb <dig> sites were identified.
moreover, most of these sites were abs sites and others located near abs sites .
the existing tsp at these loci provided additional evidence in support of historical balancing selection .
p is the number of parameters in the ω distribution, ω is the selection parameter, and pn is the proportion of sites falling into the ωn site class.
for models m <dig> and m <dig>  p and q are the shape parameters of the β function.
positively selected sites were identified in models m2a and m <dig> by the bayes empirical bayes procedure .
sites inferred under selection at the 99% level are listed in bold, and those inferred at the 95% level are shown in italics.
p is the number of parameters in the ω distribution, ω is the selection parameter, and pn is the proportion of sites falling into the ωn site class.
for models m <dig> and m <dig>  p and q are the shape parameters of the β function.
positively selected sites were identified in models m2a and m <dig> by the bayes empirical bayes procedure .
sites inferred under selection at the 99% level are listed in bold, and those inferred at the 95% level are shown in italics.
fst outlier analysis
the fst outlier analysis did not reveal a high or low level of differentiation among populations at the dqa <dig> and dqb <dig> loci compared with  <dig> microsatellite loci .
one microsatellite locus  was located outside the 95% confidence interval.
for each population, the mhc loci were also located within the 95% confidence interval of the microsatellites.
some satellites existed outside the 95% confidence interval: in the sg population, d14s <dig> was lower than the neutral level, and d1s <dig> was at the edge of the neutral level ; in the ql population, d6s <dig> was under the candidate positive selection level ; and in the snj population, d1s <dig> was above the neutral level while d6s <dig> was below .
discussion
habitat isolation generates barriers to gene flow among populations that often result in loss of genetic diversity through genetic drift and inbreeding  <cit> .
for vertebrates, genetic variation is of special importance in mhc genes due to the significant role they play in immune functions  <cit> .
mhc diversity is presumed to improve parasite resistance, reproductive success, and population viability  <cit> , and has been studied in species such as western gorilla   <cit> , brown bear   <cit> , ethiopian wolf   <cit> , european bison   <cit> , bengal tiger   <cit> , and namibian leopard   <cit> .
in this study, a relatively high level of mhc variation was found in the golden snub-nosed monkey, with  <dig> dqa <dig> and  <dig> dqb <dig> alleles found in  <dig> individuals.
however, we found lower mhc variation in the snj population, which also showed lower genetic diversity in microsatellites and mitochondrial genes  <cit> .
small sample size cannot account for the reduced variability, because sample collected across the entire distribution of the snj population showed similar patterns of genetic diversity  <cit> .
for each mhc locus, regardless of how many clones were sequenced from an individual, no more than  <dig> alleles were observed in an individual, a strong indicator that we amplified single loci in all cases.
we assumed that all alleles were from a single functional gene.
the assumption that our sequences were from functional genes was supported by three findings: 1) sites inferred to have been exposed to significant selection, most of which were abs sites, indicating historical selection at functional genes; 2) no reading frame or stop codon disruptions found in any alleles; and 3) the sequences identified from cdna were parts of or included sequences obtained from dna.
historical balancing selection
golden snub-nosed monkey mhc genes reflect historical balancing selection in that an excess of non-synonymous substitutions was mainly concentrated in the abs .
according to neutrality theory  <cit> , the synonymous nucleotide substitution rate is larger than the non-synonymous substitution rate because a change in amino acid sequence has a greater possibility of being deleterious.
the elevated rate of non-synonymous substitutions at the abs provided clear evidence of positive selection  <cit>  shaping genetic variation  <cit> .
the p-value was not significant at dqa <dig> abs may due to a weaker recombination within dqa <dig> as a lower recombination rate has been shown before.
without higher recombination and stronger selection, some dqa <dig> alleles might be lost when population size decreased.
the sharing of mhc alleles among populations also indicates that mhc alleles may have been conserved by selection  <cit> .
second, random site models analysis proved the existence of historical selection based on the maximum likelihood method, which revealed that, for mhc genes, the models including selection  match mhc alleles better than models without selection .
under the m2a and m <dig> models, some sites of the  <dig> mhc loci were under significant selection pressure.
furthermore, trans-species evolution of the mhc alleles revealed historical balancing selection.
under balancing selection, some mhc alleles or allelic lineages are reported in other species, which indicates that they are ancestral alleles  <cit> .
genes vary in terms of the level of selection, and both dqa <dig> and dqb <dig> revealed different patterns of selection.
each population had unique dqb <dig> alleles, while not every population had unique dqa <dig> alleles .
population divergence, measured as pairwise fst, was larger in dqb <dig> than in dqa <dig> except for that between sg and ql .
similar results were reported in water vole, where balancing selection pressure was different at mhc genes in continuous populations  <cit> .
mhc genes are assumed to be closely linked  <cit> .
in our study, however, linkage disequilibrium between dqa <dig> and dqb <dig> was only observed in the sg population.
this weak linkage disequilibrium and the different selection pressures on these loci could be a result of recombination, which is common at the mhc genes  <cit> .
the higher recombination rate that was found in dqb <dig> genes may explain their larger allelic richness compared to dqa <dig> genes.
recombinants maintained by selection may counteract the linkage of closely linked genes  <cit>  and play an important adaptive role in dqb <dig> evolution.
in the present study, historical selection was found, but this does not conclusively indicate that balancing selection is acting on current populations.
first, an excess of non-synonymous mutations requires a long time to accumulate  <cit> .
once present, this pattern would take a long time to vanish after the disappearance of selection  <cit> .
hence, we investigated whether selection continues to play a major role at present.
patterns of selection and drift
although selection historically maintained mhc diversity, recent population isolation and fragmentation has increased the role of genetic drift in shaping patterns of mhc variation in snub-nosed monkeys.
first, compared with neutral forces, balancing selection is supposed to diminish population differentiation as measured by conventional pairwise fst <cit> .
thus, the population structure of genes under balancing selection should not be pronounced  <cit> .
however, in the present study, half of the pairwise fst values were greater than  <dig> , and two fst values at dqb were even greater than those at microsatellites .
second, fst outlier analysis showed that the structure in the mhc loci was within the neutrality level for all populations and for each population.
considering all populations, one microsatellite  showed a fst value that was lower than the neutral level, indicating its linkage with other genes under selection  <cit> .
lastly, a positive correlation was found between allelic richness in mhc and microsatellites.
the ql population had the highest allelic richness in microsatellites and mhc, while snj had the lowest.
this positive correlation indicates that genetic drift plays a significant role in maintaining mhc diversity for snub-nosed monkeys  <cit> .
maintenance of mhc variation through balancing selection may be hampered in small, isolated populations because of their lower effective recombination rate  <cit> .
in all, our results indicate that even though selection acts on mhc, it is overwhelmed by genetic drift in small, isolated populations.
the positive correlation in the allelic richness of mhc and microsatellites, together with other evidence, indicates that genetic drift has a great influence on the maintenance of mhc variations in small, isolated populations of snub-nosed monkeys  <cit> .
no evidence showed that mhc polymorphism had increased in populations that contained low neutral variation  <cit> .
under neutral evolution theory  <cit> , alleles are expected to be neutral when s < 1/ <dig> ne .
therefore, the smaller ne becomes, the greater the likelihood of genetic drift  <cit> .
the snj population is subject to more genetic drift than the other two populations as found in a previous study  <cit> .
other animals whose patterns of mhc polymorphism have been contributed to drift over selection include the great crested newt   <cit> , black-footed rock-wallaby   <cit> , tuatara   <cit> , and the egyptian vulture   <cit> .
these results suggest that selection on mhc is not strong enough to counteract drift that results from population fragmentation, isolation and bottleneck.
CONCLUSIONS
variation at mhc loci is widely accepted as being maintained by balancing selection , even with a low level of neutral variability in some species  <cit> .
this shows the importance of balancing selection for maintaining variation in the field and exposes the problem of using neutral genes as substitutes for variation in fitness-related genes  <cit> .
however, in small, isolated populations or bottlenecked populations, balancing selection is overwhelmed by drift  <cit> .
in this study, we found the same genetic variation pattern both at neutral and mhc markers, suggesting that genetic drift was stronger than selection, thus leading to a reduction in mhc diversity in the most isolated populations.
such findings may contribute to the conservation of endangered species such as snub-nosed monkeys both in captive breeding and translocation programs.
though the relationships between mhc with mate choice and pregnancy outcomes still remain controversial  <cit> , it could be helpful to examine mhc variation in captive individuals that are involved in mating programs.
in the field, translocation of individuals from demographically and genetically healthy populations to populations that suffer from reduced genetic diversity can improve the chances of genetic recovery  <cit> .
recently, researchers have found that inbred populations could thrive with the import of migrants as part of a conservation management program  <cit> .
further, the introduction of individuals from western populations of golden snub-nosed monkeys could restore genetic diversity to the relatively homogenous snj groups, reducing the likelihood of inbreeding depression.

 understanding how levels of genetic variation influence the survival of threatened species is of fundamental interest to evolutionary and conservation biologists because many natural populations are threatened by intense reduction and fragmentation of habitat, leading to isolation, declining populations, and decreasing genetic diversity. loss of genetic diversity may increase the risk of extinction due to decreased reproductive fitness, decreased adaptive flexibility, and increased disease susceptibility. clarifying the mechanism that determines genetic variation in small, isolated populations is therefore essential for their conservation. an important assumption in conservation genetics is that small, isolated populations are more sensitive to genetic drift and inbreeding. genetic drift is the random fluctuation of allele frequencies over time; thus, adaptive alleles may be lost and deleterious alleles could be fixed in the population. the small population size and fixation of deleterious alleles leads to inbreeding depression and reduction of individual fitness, which decreases viability and compromises a population’s evolutionary adaptive potential. however, some functionally important genes that are maintained by balancing selection, such as major histocompatibility complex genes, may have a different evolutionary pattern compared with neutral markers. the multi-gene mhc family is found in vertebrates, codes for cell surface glycoproteins, and is important in animal conservation due to its role in resisting pathogens. compared to nearly neutral markers such as microsatellite loci or mitochondrial dna, which are informative for phylogenetic and phylogeographic reconstructions, mhc variability is believed to determine the capability of individuals to resist continuously evolving pathogens and parasites. consequently, mhc variability is a reflection of the processes that are related to adaptive evolution within and between populations. thus, most variation at mhc loci reflects the effects of balancing selection, which is the main mechanism for retaining high mhc genetic diversity. balancing selection includes frequency-dependent selection, overdominance and diversifying selection and promotes long evolutionary persistence of individual alleles and strongly differentiated allelic lineages in mammals. besides balancing selection, intragenic recombination has also been suggested as one evolutionary mechanism for generation of mhc sequence diversity. still, several significant questions remain. current results conflict or are unclear with regard to the relative roles of balancing selection and genetic drift in maintaining mhc polymorphism in small, isolated, or severely bottlenecked populations. studies of guppies, mexican wolves and namibian leopards found that even when genetic diversity at neutral markers was poor, polymorphisms in mhc were still maintained by balancing selection. in contrast, other research has found low levels of detectable polymorphisms for mhc genes in populations with lower diversity in neutral markers, including studies on fallow deer, northern elephant seals, great crested newt, and the black-footed rock-wallaby. these results suggest that compared to genetic drift balancing selection is relatively weak in small populations, leading to reduced variation at some mhc loci. to elucidate the role of balancing selection and genetic drift in populations, fst outlier analysis is widely used because demographic processes affect neutral loci and lead to population differentiation. the fst values are computed for all genes to distinguish the genes under selection from those non-selective genes. we studied mhc and neutral genetic variation in the golden snub-nosed monkey, an endangered primate endemic to china where it inhabits three isolated areas: sichuan and gansu provinces; the qinling mountains, shaanxi province and the shennongjia forestry district, hubei province. current census data suggest that fewer than individuals remain, and mitochondrial dna and microsatellite studies indicate that the genetically distinct snj population has very low genetic variation. the current effective population size in snj is approximately individuals. the small size and isolation that typically characterize local populations make the golden snub-nosed monkey particularly susceptible to loss of genetic variation through inbreeding and genetic drift. in this study, we 1) investigated population structure and polymorphism levels of microsatellite loci and mhc ii genes, 2) tested for both selection at the mhc loci and patterns of between population differentiations, and 3) evaluated the role of balancing selection and genetic drift in populations.  genetic variation at microsatellites genotyping of the microsatellite loci revealed that the highest level of expected heterozygosity and observed heterozygosity was in the sg population, while the lowest level was in the snj population. the hardy–weinberg equilibrium test showed no deviation from equilibrium after bonferroni correction. no linkage disequilibrium occurred for pairs of loci in any population, and micro-checker revealed no evidence of stuttering, null alleles, and allele dropout. ar, allelic richness; he, expected heterozygosity; ho, observed heterozygosity; fis, inbreeding coefficient. mhc variation, expression analyses, and recombination analysis for each locus, we analysed clones from individuals that represented three monkey populations. we obtained different dqa fragments with lengths that ranged from bp to bp including exon and partial intron and intron. after performing blast with the hla-dqa and macaca mulatta dqa exon we removed the intron and obtained dqa exon sequences with an equal length of bp. these sequences were labelled as rhro-dqa1*01– according to the nomenclature of klein et al. and were used for analysis. at the dqb locus, we identified unique dqb exon sequences that we called rhro-dqb1*01–. fragments of mhc loci were successfully amplified from the cdna of blood samples that were also used in dna amplifications. at the dqa locus, bp fragments of dqa exon were obtained from blood samples, and these fragments were part of the alleles obtained from dna in the same individual. at the dqb locus longer fragments were obtained, including the entire exon partial exon and exon 3; however, these fragments were only obtained from blood sample. shorter fragments of dqb exon were obtained from blood samples. no sequences contained stop codons. the sequences from rna were identical with that from dna, which supports the hypothesis that these mhc pcr products were transcribed and expressed. no more than two alleles were present per individual, suggesting that for each mhc locus one gene copy was sequenced with these primer sets. no stop codon or insertion/deletion was detected in dqa and dqb the measures of mhc diversity in the populations are summarized in table all three populations shared four of the nine dqa alleles and two of the among dqb alleles. the mhc sequences were highly divergent: of bp of dqa and of bp of dqb were variable. the hardy–weinberg equilibrium tests showed that the ql population deviated from equilibrium. linkage disequilibrium occurred in the sg population. population recombination analysis using ldhat revealed that the dqb locus had a higher recombination rate when all alleles were included between the mhc loci. private alleles, alleles only found in this population; h, genetic variation; ar, allele richness; fis, inbreeding coefficient. population structure and phylogenetic analysis microsatellite-based analyses detected significant genetic structures among the populations. two genetic clusters were found with a clear maximum Δk. the pairwise fst was the largest between snj and the other populations among all of the fst values, which showed that the differences between sg and ql were minimal compared to those between sg-ql and snj. however, there was also a small Δk peak at k =, and results showed that in addition to the divergence between sg and ql, there was also divergence within the sg and ql populations. these results agree with earlier studies, where the differences within the sg and ql populations resulted from their different origins and habitat fragmentation. the clear population structure observed in microsatellites was not found at either the dqa locus or the dqb locus. similar to previous studies, the relationship among mhc alleles did not agree with the geographical locations of the populations, as observed in the microsatellite results. dqa1/dqb alleles were not grouped by species but were mixed with other species. no clear clade could be identified from the phylogenetic tree at these two loci. populations had more private alleles at dqb than dqa. four among dqa exon alleles were commonly shared among the populations, while just among dqb exon alleles were shared among all of the populations. however, frequency differences of these shared alleles were found between all population pairs. comparisons of pairwise fst values of mhc and microsatellite sites are shown in figure the fst values showed lower divergence among populations compared with microsatellite loci. half of the fst values at the mhc loci were higher than, indicating that differences existed among populations, especially between sg and snj. the population difference was more distinct at the dqb locus: the differentiations between the sg and snj populations and between the ql and snj populations were even larger than the differentiation at the microsatellite. historical selection evidence for historical balancing selection was detected. first, in the abs, the number of nonsynonymous mutations was greater than the number of synonymous mutations using the z-test. although p-value was not significant for dqa locus in mega, abs sites under significant selection were detected in paml. after comparisons of various codon evolution models using codeml in paml models integrating positive selection matched mhc better than the other models based on the akaike information criterion values. under model m2a, dqa sites and dqb sites were exposed to significant selection. under model m dqa sites and dqb sites were identified. moreover, most of these sites were abs sites and others located near abs sites. the existing tsp at these loci provided additional evidence in support of historical balancing selection. p is the number of parameters in the ω distribution, ω is the selection parameter, and pn is the proportion of sites falling into the ωn site class. for models m and m p and q are the shape parameters of the β function. positively selected sites were identified in models m2a and m by the bayes empirical bayes procedure. sites inferred under selection at the 99% level are listed in bold, and those inferred at the 95% level are shown in italics. p is the number of parameters in the ω distribution, ω is the selection parameter, and pn is the proportion of sites falling into the ωn site class. for models m and m p and q are the shape parameters of the β function. positively selected sites were identified in models m2a and m by the bayes empirical bayes procedure. sites inferred under selection at the 99% level are listed in bold, and those inferred at the 95% level are shown in italics. fst outlier analysis the fst outlier analysis did not reveal a high or low level of differentiation among populations at the dqa and dqb loci compared with microsatellite loci. one microsatellite locus was located outside the 95% confidence interval. for each population, the mhc loci were also located within the 95% confidence interval of the microsatellites. some satellites existed outside the 95% confidence interval: in the sg population, d14s was lower than the neutral level, and d1s was at the edge of the neutral level; in the ql population, d6s was under the candidate positive selection level; and in the snj population, d1s was above the neutral level while d6s was below. discussion habitat isolation generates barriers to gene flow among populations that often result in loss of genetic diversity through genetic drift and inbreeding. for vertebrates, genetic variation is of special importance in mhc genes due to the significant role they play in immune functions. mhc diversity is presumed to improve parasite resistance, reproductive success, and population viability, and has been studied in species such as western gorilla, brown bear, ethiopian wolf, european bison, bengal tiger, and namibian leopard. in this study, a relatively high level of mhc variation was found in the golden snub-nosed monkey, with dqa and dqb alleles found in individuals. however, we found lower mhc variation in the snj population, which also showed lower genetic diversity in microsatellites and mitochondrial genes. small sample size cannot account for the reduced variability, because sample collected across the entire distribution of the snj population showed similar patterns of genetic diversity. for each mhc locus, regardless of how many clones were sequenced from an individual, no more than alleles were observed in an individual, a strong indicator that we amplified single loci in all cases. we assumed that all alleles were from a single functional gene. the assumption that our sequences were from functional genes was supported by three findings: 1) sites inferred to have been exposed to significant selection, most of which were abs sites, indicating historical selection at functional genes; 2) no reading frame or stop codon disruptions found in any alleles; and 3) the sequences identified from cdna were parts of or included sequences obtained from dna. historical balancing selection golden snub-nosed monkey mhc genes reflect historical balancing selection in that an excess of non-synonymous substitutions was mainly concentrated in the abs. according to neutrality theory, the synonymous nucleotide substitution rate is larger than the non-synonymous substitution rate because a change in amino acid sequence has a greater possibility of being deleterious. the elevated rate of non-synonymous substitutions at the abs provided clear evidence of positive selection shaping genetic variation. the p-value was not significant at dqa abs may due to a weaker recombination within dqa as a lower recombination rate has been shown before. without higher recombination and stronger selection, some dqa alleles might be lost when population size decreased. the sharing of mhc alleles among populations also indicates that mhc alleles may have been conserved by selection. second, random site models analysis proved the existence of historical selection based on the maximum likelihood method, which revealed that, for mhc genes, the models including selection match mhc alleles better than models without selection. under the m2a and m models, some sites of the mhc loci were under significant selection pressure. furthermore, trans-species evolution of the mhc alleles revealed historical balancing selection. under balancing selection, some mhc alleles or allelic lineages are reported in other species, which indicates that they are ancestral alleles. genes vary in terms of the level of selection, and both dqa and dqb revealed different patterns of selection. each population had unique dqb alleles, while not every population had unique dqa alleles. population divergence, measured as pairwise fst, was larger in dqb than in dqa except for that between sg and ql. similar results were reported in water vole, where balancing selection pressure was different at mhc genes in continuous populations. mhc genes are assumed to be closely linked. in our study, however, linkage disequilibrium between dqa and dqb was only observed in the sg population. this weak linkage disequilibrium and the different selection pressures on these loci could be a result of recombination, which is common at the mhc genes. the higher recombination rate that was found in dqb genes may explain their larger allelic richness compared to dqa genes. recombinants maintained by selection may counteract the linkage of closely linked genes and play an important adaptive role in dqb evolution. in the present study, historical selection was found, but this does not conclusively indicate that balancing selection is acting on current populations. first, an excess of non-synonymous mutations requires a long time to accumulate. once present, this pattern would take a long time to vanish after the disappearance of selection. hence, we investigated whether selection continues to play a major role at present. patterns of selection and drift although selection historically maintained mhc diversity, recent population isolation and fragmentation has increased the role of genetic drift in shaping patterns of mhc variation in snub-nosed monkeys. first, compared with neutral forces, balancing selection is supposed to diminish population differentiation as measured by conventional pairwise fst. thus, the population structure of genes under balancing selection should not be pronounced. however, in the present study, half of the pairwise fst values were greater than, and two fst values at dqb were even greater than those at microsatellites. second, fst outlier analysis showed that the structure in the mhc loci was within the neutrality level for all populations and for each population. considering all populations, one microsatellite showed a fst value that was lower than the neutral level, indicating its linkage with other genes under selection. lastly, a positive correlation was found between allelic richness in mhc and microsatellites. the ql population had the highest allelic richness in microsatellites and mhc, while snj had the lowest. this positive correlation indicates that genetic drift plays a significant role in maintaining mhc diversity for snub-nosed monkeys. maintenance of mhc variation through balancing selection may be hampered in small, isolated populations because of their lower effective recombination rate. in all, our results indicate that even though selection acts on mhc, it is overwhelmed by genetic drift in small, isolated populations. the positive correlation in the allelic richness of mhc and microsatellites, together with other evidence, indicates that genetic drift has a great influence on the maintenance of mhc variations in small, isolated populations of snub-nosed monkeys. no evidence showed that mhc polymorphism had increased in populations that contained low neutral variation. under neutral evolution theory, alleles are expected to be neutral when s < 1/ ne. therefore, the smaller ne becomes, the greater the likelihood of genetic drift. the snj population is subject to more genetic drift than the other two populations as found in a previous study. other animals whose patterns of mhc polymorphism have been contributed to drift over selection include the great crested newt, black-footed rock-wallaby, tuatara, and the egyptian vulture. these results suggest that selection on mhc is not strong enough to counteract drift that results from population fragmentation, isolation and bottleneck.  variation at mhc loci is widely accepted as being maintained by balancing selection, even with a low level of neutral variability in some species. this shows the importance of balancing selection for maintaining variation in the field and exposes the problem of using neutral genes as substitutes for variation in fitness-related genes. however, in small, isolated populations or bottlenecked populations, balancing selection is overwhelmed by drift. in this study, we found the same genetic variation pattern both at neutral and mhc markers, suggesting that genetic drift was stronger than selection, thus leading to a reduction in mhc diversity in the most isolated populations. such findings may contribute to the conservation of endangered species such as snub-nosed monkeys both in captive breeding and translocation programs. though the relationships between mhc with mate choice and pregnancy outcomes still remain controversial, it could be helpful to examine mhc variation in captive individuals that are involved in mating programs. in the field, translocation of individuals from demographically and genetically healthy populations to populations that suffer from reduced genetic diversity can improve the chances of genetic recovery. recently, researchers have found that inbred populations could thrive with the import of migrants as part of a conservation management program. further, the introduction of individuals from western populations of golden snub-nosed monkeys could restore genetic diversity to the relatively homogenous snj groups, reducing the likelihood of inbreeding depression. 
in this study, we examined the mechanisms maintaining polymorphisms of mhc genes in small isolated populations of the endangered golden snub-nosed monkey by comparing genetic variation found in mhc and microsatellite loci. two mhc genes were sequenced and sixteen microsatellite loci were genotyped from samples representing three isolated populations. mhc genes and microsatellite loci showed the same allelic richness pattern with the lowest genetic variation occurring in snj, suggesting that genetic drift played a prominent role in these isolated populations. as mhc genes are subject to selective pressures, the maintenance of genetic variation is of particular interest in small, long-isolated populations. small, isolated populations often experience loss of genetic variation due to random genetic drift. lowest genetic variation for both mhc and microsatellites was found in the shennongjia population. fst outlier analysis showed that population differentiation at the two mhc loci was similar to the microsatellite loci. the results of this study may contribute to captive breeding and translocation programs for endangered species. unlike neutral or nearly neutral markers, major histocompatibility complex genes in these populations may retain high levels of polymorphism due to balancing selection. the relative roles of balancing selection and genetic drift in either small isolated or bottlenecked populations remain controversial. patterns of microsatellite variation revealed population structure.
 small, isolated populations often experience loss of genetic variation due to random genetic drift. unlike neutral or nearly neutral markers, major histocompatibility complex genes in these populations may retain high levels of polymorphism due to balancing selection. the relative roles of balancing selection and genetic drift in either small isolated or bottlenecked populations remain controversial. in this study, we examined the mechanisms maintaining polymorphisms of mhc genes in small isolated populations of the endangered golden snub-nosed monkey by comparing genetic variation found in mhc and microsatellite loci. there are few studies of this kind conducted on highly endangered primate species.  two mhc genes were sequenced and sixteen microsatellite loci were genotyped from samples representing three isolated populations. we isolated nine dqa alleles and sixteen dqb alleles and validated expression of the alleles. lowest genetic variation for both mhc and microsatellites was found in the shennongjia population. historical balancing selection was revealed at both the dqa and dqb loci, as revealed by excess non-synonymous substitutions at antigen binding sites and maximum-likelihood-based random-site models. patterns of microsatellite variation revealed population structure. fst outlier analysis showed that population differentiation at the two mhc loci was similar to the microsatellite loci.  mhc genes and microsatellite loci showed the same allelic richness pattern with the lowest genetic variation occurring in snj, suggesting that genetic drift played a prominent role in these isolated populations. as mhc genes are subject to selective pressures, the maintenance of genetic variation is of particular interest in small, long-isolated populations. the results of this study may contribute to captive breeding and translocation programs for endangered species. balancing selectionconservation geneticsgene driftmhcrhinopithecus roxellana 