We found that approximately half of the SNPs identified in M. fascicularis are also present in M. mulatta. The finding indicates that efforts to identify SNPs in either species will be beneficial in generating resources for both macaque species. The recent publication of the rhesus genome sequence has sparked interest in developing genome-wide SNP arrays for use in biomedical research . Such a rhesus macaque SNP array could be used to genotype M. fascicularis DNAs, which based upon this analysis, would capture about 50% of the genomic SNPs in cynomolgus macaques.
In this sampling, we identified 36 SNPs that appeared unique to a single geographic population of M. fascicularis, suggesting that genetic differences between the populations could underlie unrecognized phenotypic differences between these animals. Since genetic heterogeneity can complicate the reproducibility of results in biomedical studies, it would be prudent to use animals from only a single population of M. fascicularis in a single research study. Towards this goal, population-specific SNPs could be used to verify the ancestry of a research animal when it is uncertain. As example, the population-specific SNPs from this study identified cynomolgus macaques from two breeding centers in China as being of Indochinese descent. An expanded SNP discovery effort would readily identify more population markers, making it possible to identify hybrid M. fascicularis as well.
The finding that approximately half of the SNPs found in M. fascicularis overlap with those found in M. mulatta is remarkable, given that previous analysis showed that only 31% of SNPs in rhesus macaques are shared by both the Indian and Chinese subpopulations [10, 11]. The overlapping SNPs in M. fascicularis include ones that are private to Chinese or Indian rhesus, as well as those that are shared between Indian and Chinese populations. While there are more SNPs from the Chinese rhesus than Indian rhesus macaques present in cynomolgus macaques, there are about twice as many SNPs present in the Chinese rhesus population as whole. An evolutionary bottleneck that reduced the overall genetic diversity of the Indian rhesus macaque has been proposed , and such a contraction could also have reduced the representation of ancestral macaque SNPs in Indian rhesus monkeys.
There are several possible explanations for the high percentage of shared variants in these two macaque species. The SNPs identified in both M. fascicularis and M. mulatta could represent that the most ancient SNPs in the fascicularis group, those that predate the divergence of rhesus and cynomolgus macaques. Consistent with this idea, the majority of SNPs found in both Indian and Chinese rhesus macaques were also found in M. fascicularis. Interestingly, although mtDNA analysis supports the divergence of these two species 1.8 MYA [32, 33], the few studies to date of nuclear DNA loci have suggested a closer relationship [29–31]. Our findings also suggest a more complex evolutionary history than that suggested by mtDNA alone.
It is possible that relatively recent gene flow between these two macaque species has contributed to the high rate of overlap between M. mulatta and M. fascicularis SNPs. The Y-chromosome sequence studies of Tosi et al. suggest interspecies hybridization, though only within the current overlapping range of Chinese rhesus macaques and Indochinese cynomolgus macaques [30, 31]. However, in this study we found that Indonesian cynomolgus macaques also share a high percentage of SNPs with the rhesus macaques. Thus if interspecies hybridization contributed to the shared sequences, it likely would have occurred before or during periods of glaciation, when land bridges could have permitted the migration of macaques as far South as Indonesia. Nonetheless, gene flow between Chinese rhesus and M. fascicularis does not explain all of the overlapping SNPs, since there is also evidence of Indian-specific rhesus variants in M. fascicularis. Due to the geographic barriers that separate India and Indonesia, SNPs common to both of these populations are more likely a consequence of either sequence conservation or convergent evolution.
Selective pressure to maintain some of the sequence variants could have contributed to the retention of some SNPs in both M. fascicularis and M. mulatta. Possible evidence of selective pressure can be found within this study. By both direct sequence comparisons and SNP array genotyping, we found that one SNP locus (CCR1:641) had a high minor allele frequency (0.41) in the macaques derived from Indochina and Chinese breeding center, and yet no heterozygous individuals were detected, a striking departure from Hardy-Weinberg equilibrium. This finding could be the consequence of inadequate sample size, or a technical issue that was resolved neither by direct sequencing nor by the SNP array. However it is also possible that a heterozygous genotype at CCR1:641, or at alleles tightly linked to this locus, is associated with decreased survival. This is not implausible, since CCR1 encodes the chemokine receptor 1 protein, which is involved in leukocyte recruitment in response to pathogenic infections .
The MAOA:116 locus has fixed alleles in the Indian and Chinese rhesus macaque populations and was included in this study of M. fascicularis. The allele present in > 99% of Indian rhesus (C) was also found in almost all of the M. fascicularis, with the only exceptions being a few individuals from the Indochinese population. This skewed presence of the Indian rhesus allele in the M. fascicularis animals is striking. The MAOA gene encodes monoamine oxidase A, a protein that is involved in the breakdown of neurotransmitters, including norepinephrine and serotonin. Some alleles of MAOA are thought to influence aggressive and impulsive behaviors in primates . Perhaps selective pressure favors different MAOA alleles in varying macaque populations or environments.
There were no fixed alleles detected in this study that distinguish M. fascicularis and M. mulatta, and thus there is no direct evidence of gene replacement between species. Based upon the morphological and anatomical differences between the macaques, one would expect some gene replacement to be present. Additional sequencing of larger regions of genomic DNA will be needed to resolve the rate of allele fixation between these two macaque species.