Predicted structural differences of four fertility-related Y-chromosome proteins in Macaca mulatta, M. fascicularis, and their Indochinese hybrids.

Species in the genus Macaca typically live in multimale-multifemale social groups with male macaques exhibiting some of the largest testis: body weight ratios among primates. Males are believed to experience intense levels of sperm competition. Several spermatogenesis genes are located on the Y-chromosome and, interestingly, occasional hybridization between two species has led to the introgression of the rhesus macaque (Macaca mulatta) Y-chromosome deep into the range of the long-tailed macaque (M. fascicularis). These observations have led to the prediction that the successful introgression of the rhesus Y-haplotype is due to functional differences in spermatogenesis genes compared to those of the native long-tailed Y-haplotype. We examine here four Y-chromosomal loci-RBMY, XKRY, and two nearly identical copies of CDY-and their corresponding protein sequences. The genes were surveyed in representative animals from north of, south of, and within the rhesus x long-tailed introgression zone. Our results show a series of non-synonymous amino acid substitutions present between the two Y-haplotypes. Protein structure modeling via I-TASSER revealed different folding patterns between the two species' Y-proteins, and functional predictions via TreeSAAP further reveal physicochemical differences as a result of non-synonymous substitutions. These differences inform our understanding of the evolution of primate Y-proteins involved in spermatogenesis and, in turn, have biomedical implications for human male fertility.

Mitonuclear interactions and introgression genomics of macaque monkeys (Macaca) highlight the influence of behaviour on genome evolution.

In most macaques, females are philopatric and males migrate from their natal ranges, which results in pronounced divergence of mitochondrial genomes within and among species. We therefore predicted that some nuclear genes would have to acquire compensatory mutations to preserve compatibility with diverged interaction partners from the mitochondria. We additionally expected that these sex-differences would have distinctive effects on gene flow in the X and autosomes. Using new genomic data from 29 individuals from eight species of Southeast Asian macaque, we identified evidence of natural selection associated with mitonuclear interactions, including extreme outliers of interspecies differentiation and metrics of positive selection, low intraspecies polymorphism and atypically long runs of homozygosity associated with nuclear-encoded genes that interact with mitochondria-encoded genes. In one individual with introgressed mitochondria, we detected a small but significant enrichment of autosomal introgression blocks from the source species of her mitochondria that contained genes which interact with mitochondria-encoded loci. Our analyses also demonstrate that sex-specific demography sculpts genetic exchange across multiple species boundaries. These findings show that behaviour can have profound but indirect effects on genome evolution by influencing how interacting components of different genomic compartments (mitochondria, the autosomes and the sex chromosomes) move through time and space.

Lowered sensitivity of bitter taste receptors to ß-glucosides in bamboo lemurs: an instance of parallel and adaptive functional decline in TAS2R16?

Bitter taste facilitates the detection of potentially harmful substances and is perceived via bitter taste receptors (TAS2Rs) expressed on the tongue and oral cavity in vertebrates. In primates, TAS2R16 specifically recognizes ß-glucosides, which are important in cyanogenic plants' use of cyanide as a feeding deterrent. In this study, we performed cell-based functional assays for investigating the sensitivity of TAS2R16 to ß-glucosides in three species of bamboo lemurs (Prolemur simus, Hapalemur aureus and H. griseus), which primarily consume high-cyanide bamboo. TAS2R16 receptors from bamboo lemurs had lower sensitivity to ß-glucosides, including cyanogenic glucosides, than that of the closely related ring-tailed lemur (Lemur catta). Ancestral reconstructions of TAS2R16 for the bamboo-lemur last common ancestor (LCA) and that of the Hapalemur LCA showed an intermediate sensitivity to ß-glucosides between that of the ring-tailed lemurs and bamboo lemurs. Mutagenetic analyses revealed that P. simus and H. griseus had separate species-specific substitutions that led to reduced sensitivity. These results indicate that low sensitivity to ß-glucosides at the cellular level-a potentially adaptive trait for feeding on cyanogenic bamboo-evolved independently after the Prolemur-Hapalemur split in each species.

Mitogenomics of macaques (Macaca) across Wallace's Line in the context of modern human dispersals.

Wallace's Line demarcates a biogeographical boundary between the Indomalaya and Australasian ecoregions. Most placental mammalian genera, for example, occur to the west of this line, whereas most marsupial genera occur to the east. However, macaque monkeys are unusual because they naturally occur on both western and eastern sides. To further explore this anomalous distribution, we analyzed 222 mitochondrial genomes from ∼20 macaque species, including new genomes from 60 specimens. These comprise a population sampling of most Sulawesi macaques, Macaca fascicularis (long-tailed macaques) specimens that were collected by Alfred R. Wallace and specimens that were recovered during archaeological excavations at Liang Bua, a cave on the Indonesian island of Flores. In M. fascicularis, three mitochondrial lineages span the southernmost portion of Wallace's Line between Bali and Lombok, and divergences within these lineages are contemporaneous with, and possibly mediated by, past dispersals of modern human populations. Near the central portion of Wallace's Line between Borneo and Sulawesi, a more ancient dispersal of macaques from mainland Asia to Sulawesi preceded modern human colonization, which was followed by rapid dispersal of matrilines and was subsequently influenced by recent interspecies hybridization. In contrast to previous studies, we find no strong signal of recombination in most macaque mitochondrial genomes. These findings further characterize macaque evolution before and after modern human dispersal throughout Southeast Asia and point to possible effects on biodiversity of ancient human cultural diasporas.

Structural variations of subterminal satellite blocks and their source mechanisms as inferred from the meiotic configurations of chimpanzee chromosome termini.

African great apes have large constitutive heterochromatin (C-band) blocks in subtelomeric regions of the majority of their chromosomes, but humans lack these. Additionally, the chimpanzee meiotic cell division process demonstrates unique partial terminal associations in the first meiotic prophase (pachytene). These are likely formed as a result of interaction among subtelomeric C-band blocks. We thus conducted an extensive study to define the features in the subtelomeric heterochromatic regions of chimpanzee chromosomes undergoing mitotic metaphase and meiotic cell division. Molecular cytogenetic analyses with probes of both subterminal satellite DNA (a main component of C-band) and rDNA demonstrated principles of interaction among DNA arrays. The results suggest that homologous and ectopic recombination through persistent subtelomeric associations (post-bouquet association observed in 32% of spermatocytes in the pachytene stage) appears to create variability in heterochromatin patterns and simultaneously restrain subtelomeric genome polymorphisms. That is, the meeting of non-homologous chromosome termini sets the stage for ectopic pairing which, in turn, is the mechanism for generating variability and genomic dispersion of subtelomeric C-band blocks through a system of concerted evolution. Comparison between the present study and previous reports indicated that the chromosomal distribution rate of sutelomeric regions seems to have antagonistic correlation with arm numbers holding subterminal satellite blocks in humans, chimpanzees, and gorillas. That is, the increase of subterminal satellite blocks probably reduces genomic diversity in the subtelomeric regions. The acquisition vs. loss of the subtelomeric C-band blocks is postulated as the underlying engine of this chromosomal differentiation yielded by meiotic chromosomal interaction.

Diversifying selection detected in only a minority of xenobiotic-metabolizing CYP1-3 genes among primate species.

1. Primates exhibit a high degree of among-species dietary diversity, which likely exposes them to varying levels of xenobiotic compounds. Here, we examined the evolution of primate CYP1-3 gene families, and we classified the 15 CYP1-3 gene subfamilies as either xenobiotic-metabolizing (XM) or endogenous-metabolizing (EM) based on sources in the P450 literature. 2. We predicted that XM P450s would show (1) greater variability in gene-copy number and (2) more evidence of diversifying selection and, especially on codons that encode the substrate-recognition sites (SRSs) for the final enzymes. 3. Counter to our first prediction, EM and XM P450s showed similar levels of variation in gene-copy number. We did find, however, that four XM P450 subfamilies (CYP2C, CYP2D, CYP2E, and CYP3A) showed evidence of diversifying selection while no EM subfamilies demonstrated any consistent signal of diversifying selection. Of these four, CYP2C, CYP2D, and CYP3A showed significant links between SRSs and diversifying selection. 4. These results reveal an amount of evolutionary dynamism that would not be expected when viewing P450 subfamilies along a simple binary EM/XM spectrum. We recommend that comparative studies of cytochrome P450 evolution should focus on the CYP2C, CYP2D, CYP2E, and CYP3A subfamilies.

Evolution of Acidic Mammalian Chitinase Genes (CHIA) Is Related to Body Mass and Insectivory in Primates.

Insects are an important food resource for many primates, but the chitinous exoskeletons of arthropods have long been considered to be indigestible by the digestive enzymes of most mammals. However, recently mice and insectivorous bats were found to produce the enzyme acidic mammalian chitinase (AMCase) to digest insect exoskeletons. Here, we report on the gene CHIA and its paralogs, which encode AMCase, in a comparative sample of nonhuman primates. Our results show that early primates likely had three CHIA genes, suggesting that insects were an important component of the ancestral primate diet. With some exceptions, most extant primate species retain only one functional CHIA paralog. The exceptions include two colobine species, in which all CHIA genes have premature stop codons, and several New World monkey species that retain two functional genes. The most insectivorous species in our sample also have the largest number of functional CHIA genes. Tupaia chinensis and Otolemur garnettii retain three functional CHIA paralogs, whereas Tarsius syrichta has a total of five, two of which may be duplications specific to the tarsier lineage. Selection analyses indicate that CHIA genes are under more intense selection in species with higher insect consumption, as well as in smaller-bodied species (<500 g), providing molecular support for Kay's Threshold, a well-established component of primatological theory which proposes that only small primates can be primarily insectivorous. These findings suggest that primates, like mice and insectivorous bats, may use the enzyme AMCase to digest the chitin in insect exoskeletons, providing potentially significant nutritional benefits.

Considerable Synteny and Sequence Similarity of Primate Chromosomal Region VIIq31.

Human chromosome 7 has been the focus of many behavioral, genetic, and medical studies because it carries genes related to cancer and neurodevelopment. We examined the evolution of the chromosome 7 homologs, and the 7q31 region in particular, using chromosome painting analyses and 3 paint probes derived from (i) the whole of chimpanzee chromosome VII (wcVII), (ii) human 7q31 (h7q31), and (iii) the chimpanzee homolog VIIq31 (cVIIq31). The wcVII probe was used instead of the whole human chromosome 7 because the chimpanzee contains additional C-bands and revealed large areas of synteny conservation as well as fragmentation across 20 primate species. Analyses focusing specifically on the 7q31 homolog and vicinity revealed considerable conservation across lineages with 2 exceptions. First, the probes verified an insertion of repetitive sequence at VIIq22 in chimpanzees and bonobos and also detected the sequence in most subtelomeres of the African apes. Second, a paracentric inversion with a breakpoint in the cVIIq31 block was found in the common marmoset, confirming earlier studies. Subsequent in silico comparative genome analysis of 17 primate species revealed that VIIq31.1 is more significantly conserved at the sequence level than other regions of chromosome VII, which indicates that its components are likely responsible for critical shared traits across the order, including conditions necessary for proper human development and wellbeing.

Retained duplications and deletions of CYP2C genes among primates.

The human genome encodes about 60 functional enzymes of the cytochrome P450 superfamily, including four functional enzymes of the cytochrome P450 2C (CYP2C) subfamily. These enzymes have been shown to metabolize drugs and xenobiotic toxins, such as those in the diet, and are therefore of great importance for biomedical research and applications. While the pharmacology of P450 enzymes has been studied extensively, our understanding of molecular evolution of this gene family is incomplete, in part because a great variation exists in the number of CYP2C homologs across genomes. In humans, the enzymes encoded by these genes are responsible for the metabolism of more than 20% of clinical drugs, but this is not the naturalistic function of these enzymes, which is the metabolism of xenobiotics such as plant secondary metabolites. In this paper, we sought to correlate evolutionary relationships among primate CYP2C genes with known dietary profiles from these species, testing the hypothesis that these genes have evolved under the pressure of dietary toxins. Aside from a small number of deeply divergent genes, primate CYP2C paralogs form three separate clades: CYP2C18, CYP2C9/CYP2C19, and CYP2C8/CYP2C20. Our results showed that the CYP2C18 gene has been separately lost in Nomascus leucogenys and the Panini genomes, and there is no evidence that this gene has been under any positive selection among primates. While CYP2C20 has been retained in cercopithecoids, orthologous loci were separately lost in platyrrhines and hominoids. Notably, nine codons exhibited signature of positive selection. Finally, the CYP2C19 locus was duplicated in basal catarrhines, resulting in the birth of CYP2C9; but the ancestral locus was only retained in hominoid taxa. Overall, our findings support the hypothesis that primate CYP2C genes have evolved in response to selective pressures provided by dietary toxins, although not all gene clusters have evolved in the same manner. Our results may indicate an evolutionarily deep difference in ecology or physiology among higher-order primate taxa.

X Chromosome Introgression and Recombination in the cephus Group of Cercopithecus Monkeys.

A representative of Cercopithecus erythrotis was surveyed at a 9.3-kb region of the X chromosome. The data were compared against homologous sequences of closely related Cercopithecus monkeys including C. cephus, a species recently shown to have 2 polymorphic X-chromosomal lineages. Direct sequence comparisons and subsequent phylogenetic analyses revealed that synapomorphies in the first 4.3 kb cluster C. erythrotis with one C. cephus lineage, while synapomorphies in the latter 5.0 kb join it with the second C. cephus lineage. This pattern very likely reflects an ancestral episode of introgression from C. cephus into C. erythrotis followed by a recombination event. Similar groups of synapomorphies occur at different phylogenetic depths within the C. erythrotis/C. cephus/C. ascanius radiation and reveal new details in the evolutionary history of this 3-species clade.

The efficacy of sex-chromosomal markers in studies of Cercopithecus hybridization: Discovery of a captive hybrid and applications in wild populations.

We demonstrate the utility of previously described molecular methods for identifying hybrid Cercopithecus monkeys. Using phylogenetic analyses and DNA sequence comparisons at X-chromosomal and Y-chromosomal loci, we have identified a hybrid animal in the Cheyenne Mountain Zoo (USA)--an identification that was not known a priori but was later confirmed by review of zoo records. The molecular techniques employed here are of great use to studies of the genus Cercopithecus because, unlike most mammals, these monkeys frequently form polyspecific associations, and recent deforestation is likely to have driven otherwise low-level hybridization to higher frequencies which may reduce the fitness of threatened populations. Y-chromosomal markers are especially informative because they provide working hypotheses for (1) the primary mechanism of hybridization (i.e., species A males × species B females) and, by extension; (2) the major direction of gene flow.

Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis).

BACKGROUND: Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon's range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing. RESULTS: Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma. CONCLUSIONS: Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.

Molecular histology of spermatogenesis in the Japanese macaque monkey (Macaca fuscata).

Non-human primates are our closest relatives and therefore offer valuable comparative models for human evolutionary studies and biomedical research. As such, Japanese macaques (Macaca fuscata) have contributed to the advancement of primatology in both field and laboratory settings. Specifically, Japanese macaques serve as an excellent model for investigating postnatal development and seasonal breeding in primates because of their relatively prolonged juvenile period and distinct seasonal breeding activity in adulthood. Pioneering histological studies have examined the developmental associations between their reproductive states and spermatogenesis by morphological observation. However, a molecular histological atlas of Japanese macaque spermatogenesis is only in its infancy, limiting our understanding of spermatogenesis ontogeny related to their reproductive changes. Here, we performed immunofluorescence analyses of spermatogenesis in Japanese macaque testes to determine the expression of a subset of marker proteins. The present molecular histological analyses readily specified major spermatogonial subtypes as SALL4+ A spermatogonia and Ki67+/C-KIT+ B spermatogonia. The expression of DAZL, SCP1, γH2AX, VASA, and calmegin further showed sequential changes regarding the protein expression profile and chromosomal structures during spermatogenesis in a differentiation stage-specific manner. Accordingly, comparative analyses between subadults and adults identified spermatogenic deficits in differentiation and synchronization in subadult testes. Our findings provide a new diagnostic platform for dissecting spermatogenic status and reproduction in the Japanese macaques.

Monoamine oxidase polymorphisms in rhesus and Japanese macaques (Macaca mulatta and M. fuscata).

Monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) are enzymes that degrade several monoamines of the central nervous system and have long been implicated in the modulation of social behavior. Macaque monkeys are a suitable model for investigating the role of functional monoamine oxidase polymorphisms in behavior modulation given the high amount of social diversity among the nearly two dozen species. The present study reports allele frequencies for two polymorphisms, MAOA-LPR and MBin2, in samples of rhesus (Macaca mulatta) and Japanese (M. fuscata) macaques. Our results suggest that the two species may differ in high- and low-activity MAOA-LPR allele frequencies. Specifically, 89% of the Japanese macaque alleles in our sample were the low-activity variant, whereas only 41% of the rhesus macaque alleles were of this sort. In our samples, the two species possessed similar allelic variation at the MBin2 locus, with each possessing some species-specific alleles. We also tested for associations between MAOA-LPR genotype and plasma serotonin (5-HT) and dopamine (DA) concentrations in a subset of rhesus macaques, which revealed no association with genotype. Our findings point toward potential differences in the monoaminergic system of two closely related macaque species. Discussion of our results are centered on implications for future investigations that aim to better understand the functionality of monoamine oxidase polymorphisms in the context of primate social behavior.

Mitochondrial evidence for the hybrid origin of the kipunji, Rungwecebus kipunji (Primates: Papionini).

Common baboons (Papio), gelada baboons (Theropithecus) and baboon-mangabeys (Lophocebus) are closely related African papionin monkeys. In 2005, the species Lophocebus kipunji was described from relict montane and submontane forests in Tanzania, based upon a single specimen and observations of living animals. Its initial assignment to Lophocebus was based on its overall morphology, but subsequent genetic studies suggesting that it was sister taxon to common baboons (Papio) led to its generic separation, as Rungwecebus. As a mangabey-like sister-taxon to Papio, Rungwecebus could be interpreted either as an arboreal derivative from a more terrestrial, baboon-like ancestor, or as a survivor of a mangabey-like common ancestor of the Lophocebus-Papio-Theropithecus clade. Here, we present a new, strongly-supported, mitochondrial DNA (mtDNA) phylogeny that includes Papio baboons from populations geographically close to the kipunji. Rather than supporting sister-taxon status, the new phylogeny not only situates the kipunji's mtDNA among Papio haplotypes, it clearly assigns it to a mitochondrial clade including geographically adjacent yellow baboons (Papio cynocephalus). This relationship suggests either that the kipunji is descended from a yellow baboon, and has converged on a mangabey-like morphology, or, much more likely, that it originated by hybridization between Papio cf.cynocephalus females and Lophocebus sp. males, about 0.65 Ma. We believe this to be the first case among mammals in which a natural occurrence of inter-generic hybridization can be shown to have resulted in a new, distinct, long-surviving taxon. More such cases can be anticipated as molecular evidence accumulates.

A mobile element-based evolutionary history of guenons (tribe Cercopithecini).

BACKGROUND: Guenons (tribe Cercopithecini) are a species-rich group of primates that have attracted considerable attention from both primatologists and evolutionary biologists. The complex speciation pattern has made the elucidation of their relationships a challenging task, and many questions remain unanswered. SINEs are a class of non-autonomous mobile elements and are essentially homoplasy-free characters with known ancestral states, making them useful genetic markers for phylogenetic studies. RESULTS: We identified 151 novel Alu insertion loci from 11 species of tribe Cercopithecini, and used these insertions and 17 previously reported loci to infer a phylogenetic tree of the tribe Cercopithecini. Our results robustly supported the following relationships: (i) Allenopithecus is the basal lineage within the tribe; (ii) Cercopithecus lhoesti (L'Hoest's monkey) forms a clade with Chlorocebus aethiops (African green monkey) and Erythrocebus patas (patas monkey), supporting a single arboreal to terrestrial transition within the tribe; (iii) all of the Cercopithecus except C. lhoesti form a monophyletic group; and (iv) contrary to the common belief that Miopithecus is one of the most basal lineages in the tribe, M. talapoin (talapoin) forms a clade with arboreal members of Cercopithecus, and the terrestrial group (C. lhoesti, Chlorocebus aethiops and E. patas) diverged from this clade after the divergence of Allenopithecus. Some incongruent loci were found among the relationships within the arboreal Cercopithecus group. Several factors, including incomplete lineage sorting, concurrent polymorphism and hybridization between species may have contributed to the incongruence. CONCLUSION: This study presents one of the most robust phylogenetic hypotheses for the tribe Cercopithecini and demonstrates the advantages of SINE insertions for phylogenetic studies.

Medical-grade buccal swabs versus drugstore cotton swabs: No difference in DNA yield.

We tested three types of medical-grade buccal swabs against standard cotton swabs for differences in DNA yield. A panel of swab types - one drugstore (Q-tips ® ) and three medical-grade - was used for buccal cell collection from three different individuals. DNA was extracted from all swabs using a QIAcube robot; quantitation values were measured by an Alu-based qPCR assay; and differences were compared through a 2-way ANOVA. Our results demonstrate that cotton swabs recover as much DNA as medical-grade swabs, but at a tremendously lower cost. Cotton swabs also display the greatest consistency of DNA yield, as indicated by the lowest coefficient of variation among the four tested swab types. These findings suggest that the use of standard cotton swabs for buccal cell collection offers not only a significant cost savings, but a more consistent method compared to the use of medical-grade swabs.

Correction to 'Speciation over the edge: gene flow among non-human primate species across a formidable biogeographic barrier'.

[This corrects the article DOI: 10.1098/rsos.170351.].

Speciation over the edge: gene flow among non-human primate species across a formidable biogeographic barrier.

Many genera of terrestrial vertebrates diversified exclusively on one or the other side of Wallace's Line, which lies between Borneo and Sulawesi islands in Southeast Asia, and demarcates one of the sharpest biogeographic transition zones in the world. Macaque monkeys are unusual among vertebrate genera in that they are distributed on both sides of Wallace's Line, raising the question of whether dispersal across this barrier was an evolutionary one-off or a more protracted exchange-and if the latter, what were the genomic consequences. To explore the nature of speciation over the edge of this biogeographic divide, we used genomic data to test for evidence of gene flow between macaque species across Wallace's Line after macaques colonized Sulawesi. We recovered evidence of post-colonization gene flow, most prominently on the X chromosome. These results are consistent with the proposal that gene flow is a pervasive component of speciation-even when barriers to gene flow seem almost insurmountable.

Paternal, maternal, and biparental molecular markers provide unique windows onto the evolutionary history of macaque monkeys.

We report the results of one of the first intrageneric analyses to simultaneously survey mitochondrial, Y-chromosomal, and autosomal loci from the same individuals representing the same taxa. Phylogenetic trees were constructed for each of these genetic systems from a pool of 63 macaques, representing all 19 recognized species in this genus, and eight outgroup taxa. The mitochondrial locus analyzed here (1.5 Kb) spans the 3' end of 12S rDNA, tRNA-VAL, and the 5' end of 16S rDNA; the Y chromosome dataset (3.1 Kb) consists of the genes SRY and TSPY; the two autosomal datasets include IRBP intron 3 (1.6 Kb) and the 5' half of C4 "long" intron 9 (3.3 Kb). A total of 1.35 million bases were read, revealing 682 variable sites within the genus Macaca. With regard to earlier unresolved issues of macaque evolution, a comparison of topologies reconstructed from each of the three genetic systems suggests: (1) four monophyletic species groups; (2) an initial bifurcation among Asian macaques between the silenus group progenitor and a M. fascicularis-like taxon, with the latter representing the probable common ancestor to all non-silenus group Asian macaques; (3) a possible hybrid origin of M. arctoides from proto-M. assamensis/thibetana and proto-M. fascicularis; and (4) contemporary introgression between M. mulatta and M. fascicularis in Indochina. Inferences 3 and 4 are of particular interest, because episodes of reticulate evolution often go undetected in analyses employing a single genetic system. Finally, divergence calculations suggest that, in female-philopatric taxa, mitochondrial bifurcations may typically predate Y-chromosomal divergences at the same node.