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. 2023 Oct 12;40(11):msad229. doi: 10.1093/molbev/msad229

Phylogenomics Reveals High Levels of Incomplete Lineage Sorting at the Ancestral Nodes of the Macaque Radiation

Xinxin Tan 1,2,#, Jiwei Qi 3,#, Zhijin Liu 4,#, Pengfei Fan 5, Gaoming Liu 6, Liye Zhang 7, Ying Shen 8, Jing Li 9, Christian Roos 10,11,✉, Xuming Zhou 12,✉, Ming Li 13,✉,b
Editor: Jun Gojobori
PMCID: PMC10638670  PMID: 37823401

Abstract

The genus Macaca includes 23 species assigned into 4 to 7 groups. It exhibits the largest geographic range and represents the most successful example of adaptive radiation of nonhuman primates. However, intrageneric phylogenetic relationships among species remain controversial and have not been resolved so far. In this study, we conducted a phylogenomic analysis on 16 newly generated and 8 published macaque genomes. We found strong evidence supporting the division of this genus into 7 species groups. Incomplete lineage sorting (ILS) was the primary factor contributing to the discordance observed among gene trees; however, we also found evidence of hybridization events, specifically between the ancestral arctoides/sinica and silenus/nigra lineages that resulted in the hybrid formation of the fascicularis/mulatta group. Combined with fossil data, our phylogenomic data were used to establish a scenario for macaque radiation. These findings provide insights into ILS and potential ancient introgression events that were involved in the radiation of macaques, which will lead to a better understanding of the rapid speciation occurring in nonhuman primates.

Keywords: Macaca, phylogenomics, incomplete lineage sorting, hybridization, radiation

Introduction

The development of high-throughput sequencing technology and new computational tools for phylogenomics has enabled researchers to reconstruct the evolutionary histories of organisms by analyzing the distribution of inherited genetic markers in descendant lineages and looking back in time to identify those shared by a common ancestor (Figueiró et al. 2017; Árnason et al. 2018; Steenwyk et al. 2023). “Phylogenetic trees” generated from genomic data represent the most complete and best-supported hypotheses for the evolutionary history of a given organism; however, phylogenomic studies regularly reveal conflicting tree topologies, making it difficult to resolve certain branches in the tree of life (Whelan et al. 2017). Incomplete lineage sorting (ILS) is one potential explanation for gene tree discordances. ILS is primarily observed during rapid speciation events, when new lineages descend within a short time period from ancestors, particularly when there is a large effective population size (Ne) (Maddison and Knowles 2006; Avise and Robinson 2008). As a result, ILS causes ancestral polymorphisms to persist, even when descendant lineages have diverged (Szöllősi et al. 2015). Introgression and hybridization, however, are alternative explanations for gene tree discordances. Over the past decade, their role in evolutionary diversification has gained significant interest (Roos et al. 2011; Zinner et al. 2011; Abbott et al. 2013; Tung and Barreiro 2017; Fontsere et al. 2019; Van der Valk et al. 2020; Vanderpool et al. 2020). Hybridization is one of the most interesting topics in evolutionary biology. Genetic introgression caused by hybridization is not always maladaptive and may represent a potent evolutionary force (Abi-Rached et al. 2011; Abbott et al. 2013; Abbott et al. 2016; Zheng et al. 2020). Therefore, both ILS and gene flow or hybridization may result in topological incongruences between gene trees (the term “gene tree” is frequently used as shorthand for any locus in the genome, such as a protein-coding or protein-noncoding region) and species trees (Heled and Drummond 2010; Feng et al. 2022). For example, Edelman et al. (2019) used 20 de novo genome assemblies to explore speciation and gene flow in rapidly radiating Heliconius butterflies to distinguish ILS from introgression. They found that introgressed loci are underrepresented in low-recombination and gene-rich regions (Edelman et al. 2019). For primates, data for a genome-wide view on hybridization remain limited, but genome sequencing efforts are underway (Wu et al. 2022). For example, Rogers et al. (2019) and Sørensen et al. (2023) revealed complex reticulation in baboons and documented multiple episodes of admixture and introgression during the radiation of Papio using a comparative genomic approach.

The genus Macaca (Primates: Cercopithecidae) exhibits the largest geographic range and represents the most successful example of adaptive radiation of nonhuman primates (Zinner et al. 2013) (Fig. 1a). The 23 currently recognized macaque species may be subdivided into 4 to 7 groups according to differences in morphology, ecology, behavior, distribution, and genetics (Fooden 1976; Delson 1980; Morales and Melnick 1998; Groves 2001; Tosi, Disotell, et al. 2003; Sinha et al. 2005; Chakraborty et al. 2007; Zinner et al. 2013; Roos et al. 2014; Li et al. 2015; Hou et al. 2016; Fan et al. 2017; Roos et al. 2019) (Supplementary Fig. S1). However, the number and composition of these groups is controversial. Before the whole-genome area, phylogenetic analyses were primarily based on a few mitochondrial and/or nuclear markers, which resulted in contradicting phylogenetic relationships (Tosi et al. 2000; Tosi, Disotell, et al. 2003; Tosi, Morales, and Melnick 2003; Li, Handsaker, et al. 2009; Li, Han, et al. 2009; Fan et al. 2018). They were partially caused by introgression and hybridization events occurring among various macaque species.

Fig. 1.

Fig. 1.

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Macaque phylogenetics and differentiation. a) Distribution ranges of the 16 macaque species examined in this study. The range overlaps are shown in blended colors. b) An MSC species tree was constructed based on 6,351 individual GFs. Branches with a low q1 (<0.8) are numbered 1 to 6. All branches received maximal support (P = 1.0, ASTRAL analysis). Branch lengths were calculated from an ML analysis. Different species groups are represented by different background colors, which are show to the left. c) ASTRAL quartet-score analyses for branches 1 to 6 in b). Quartet scores were calculated for the 3 possible arrangements (q1 to q3) for the respective branches. The principal quartet trees are depicted, with q1 representing the species tree. Two alternative topologies for branch no. 2 as shown in c). d) Genealogical discordance across the genome of the rhesus macaque as demonstrated by a window analysis (200-kb window size) of a full-genome alignment mapped to Panu_3.0 (chromosomes shown at the bottom). The y-axis indicates the percentage of windows within a given interval (every 5 windows next to each other) that conform to (red) or reject (blue and gray) the species tree. e) Consensus network for macaques based on 6,351 GFs with a minimum threshold of 30% to form an edge. f) Distribution of FST within and between different macaque species groups (P = 0.05, Wilcoxon test).

Introgression and hybridization events between macaque species are common, for instance, among Sulawesi macaques, between Macaca mulatta and Macaca fascicularis, and between M. mulatta and Macaca thibetana (Ciani et al. 1989; Evans et al. 2001, 2003; Huson and Bryant 2006; Yan et al. 2011; Hamada et al. 2012; Fan et al. 2014; Evans et al. 2017; Ito et al. 2020). Moreover, for Macaca arctoides, a hybrid origin was suggested as different molecular markers placed the species into different clades (Tosi et al. 2000; Tosi, Morales, and Melnick 2003; Li, Handsaker, et al. 2009; Li, Han, et al. 2009; Fan et al. 2018; Roos et al. 2019). Most analyses, however, have focused on gene flow within macaque species groups or between species of the fascicularis/mulatta and arctoides/sinica species groups. Few studies include samples from all species groups for the identification of genus-wide hybridization and few attempted to identify ancient hybridization events in macaques (Fan et al. 2018; Matsudaira et al. 2018). A recent study described the history of the genus Macaca using 14 individuals from 9 species and detected extensive gene flow signals, with the strongest signals occurring between the fascicularis/mulatta and silenus groups (Song et al. 2021). Introgression signals among island species, such as Sulawesi macaques and other species, were also observed. This put forward a variety of possible reasons for this phenomenon including the genomic similarity of closely related species or ancestral introgression. Therefore, whether hybridization occurred among all macaques, especially among species that are geographically distant, still needs to be investigated. Furthermore, it remains unclear how much ancient hybridization or ILS has contributed to the diversity of the macaque genome.

In this study, we traced the complex, likely reticulated evolutionary history of macaques and newly sequenced whole genomes of 16 individuals representing 13 Asian species (M. mulatta, Macaca cyclopis, Macaca fuscata, M. arctoides, Macaca assamensis, Macaca leucogenys, Macaca sinica, Macaca leonina, Macaca silenus, Macaca nemestrina, Macaca maura, Macaca tonkeana, and Macaca nigra) and the only African macaque species (Macaca sylvanus). The sequences were mapped to the Papio anubis reference genome (Panu_3.0) to obtain single nucleotide variants (SNVs). Combined with 8 published macaque genomes, we performed a comprehensive genomics analysis of the 24 macaque individuals representing 16 species and all species groups. We reconstructed the phylogeny of the genus Macaca, investigated ILS and hybridization among the macaque species, and traced their evolutionary history and rapid radiation.

Results

Genome Sequencing and Variant Discovery

Whole-genome sequencing was performed using the Illumina HiSeq 2500 and 4000 platforms. Greater than 1,364 Gb of clean data (quality-controlled reads) were obtained, and the statistical results of the clean data for each species are listed in Supplementary Table S1. Published genome data for 8 macaques (M. mulatta [Chinese rhesus macaque, CR], M. mulatta [Indian rhesus macaque, IR], M. fascicularis, M. arctoides, M. assamensis, M. thibetana, M. nemestrina, and M. tonkeana) were downloaded from NCBI (Supplementary Table S1). Clean data for each species were mapped to the P. anubis reference genome (Panu_3.0) and yielded a genome-wide coverage of 16.85 to 42.34× with an average effective depth of 28.24× (Supplementary Table S1). We selected a more distantly related reference genome (P. anubis), rather than the rhesus macaque reference genome, to avoid any bias that may arise from mapping to an in-group reference genome. Although this may result in some data loss (in regions that lack orthologs in P. anubis), the mapping coverage (4×) for macaques ranges from 93.55% to 97.59%, which is very similar to the mapping coverage (4×) for Papio (98.75%), suggesting limited bias. The mapping rate for M. nemestrina (2) was less than 90%, which is likely the result of low DNA quality.

The number of high-quality SNVs for each individual ranged from 15,943,244 to 21,778,812, which included both homozygous and heterozygous alternatives (Supplementary Table S2). Genome-wide heterozygosity varied considerably among the macaques (Supplementary Fig. S2 and Table S2), which may partially reflect different demographic histories. Based on gene annotations from the reference genome, the exonic SNVs in macaques ranged from 225,336 to 257,712 (Supplementary Table S3). The species-specific SNVs for each macaque ranged from 1,279,649 to 8,688,344 (Supplementary Table S2).

Phylogeny of Macaques

To reconstruct the phylogeny of macaques, we generated consensus sequences for genomes from all macaques. We split the aligned chromosomes into 6,351 nonoverlapping genome fragments (GFs) of 200 kb each, which represented 46% of the genome sequence. The multispecies coalescent (MSC) species tree based on the 6,351 GFs was supported with posterior probabilities (PPs) of 1.0 for all branches (Fig. 1b; Supplementary Fig. S3). This species tree, consistent with a neighbor-joining (NJ) tree and a maximum-likelihood (ML) tree based on autosomal SNVs (Supplementary Figs. S4 and S5), supported the segregation of macaques into 7 well-supported clades or lineages that corresponded to the classification of the genus into 7 species groups (Zinner et al. 2013; Roos et al. 2014). Among them, the African sylvanus lineage diverged first, followed by an initial separation of extant Asian macaques into a clade containing the silenus and nigra lineages and a clade including all other Asian lineages. The latter segregated into a clade including the arctoides and sinica lineages and another clade containing the fascicularis and mulatta lineages (Fig. 1b; Supplementary Figs. S3 to S5). The ML tree based on the mitochondrial genomes (mitogenomes), consistent with previous results (Fan et al. 2017; Roos et al. 2019), revealed that among the Asian macaques, the silenus/nigra clade separated first, followed by the sinica lineage. Of the remaining lineages, fascicularis split first, whereas mulatta and arctoides split afterwards (Supplementary Fig. S6 and Table S4). The quartet score “q,” which is a support value for possible phylogenetic arrangements, revealed a conflict in resolving the branch leading to the ancestor of the fascicularis/mulatta and arctoides/sinica groups (Fig. 1c and d, branch no. 2 in Fig. 1b; Supplementary Fig. S3).

A consensus network analysis of the GF trees revealed a network with bifurcating branches that emerged from a central cycle in the center of the network, indicating conflicting signals for the position of the silenus/nigra clade among Asian macaques (Fig. 1e; Supplementary Fig. S7). At a threshold of 30% for conflicting edges, the position of the silenus/nigra, arctoides/sinica, and fascicularis/mulatta clades could not be ascertained (Supplementary Fig. S7). When the threshold for conflicting edges was reduced, the phylogenetic signal was even more complex, indicating additional phylogenetic conflict (Supplementary Fig. S7). FST analyses suggested significant differences within (medians for FST = 0.199) and between (medians for FST = 0.467) the different macaque groups (P < 0.0001) (Supplementary Table S5 and Fig. S1f). A clear division of macaques into different clusters was also revealed by principal component analysis (PCA) (Supplementary Fig. S8 and Table S6).

Contributions of ILS to Gene Tree Heterogeneity

The topology cluster indicated that these 2 alternative topologies are the most common among all 6,351 GF trees (Figs. 1c and 2a and b). Our study aimed to investigate the extent to which the levels of gene tree heterogeneity observed in macaques may be attributed to ILS, particularly with respect to the phylogenetic conflict among the fascicularis/mulatta, arctoides/sinica, and silenus/nigra clades (Figs. 1c and 2a and b). To differentiate between ILS and introgression, we performed QuIBL, a recently developed tree-based method introduced by Edelman et al. (2019). QuIBL estimates the internal branch length distribution in discordant topologies for triplets of species and then calculates the likelihood that this distribution is consistent with either ILS and introgression or ILS alone. Using 3,175 trees (20-kb windows separated by 400-kb windows), we observed that among members of the fascicularis/mulatta, silenus/nigra, and sinica/arctoides clades, 73 of 120 tested triplets (60.8%) with internal branches exhibited phylogenetic discordances solely due to ILS (ΔBayesian information criterion [BIC] > 10). Furthermore, only 11 of the 120 triplets (9.1%) showed strong evidence of introgression (ΔBIC < −10) (Supplementary Table S7). For example, using QuIBL on the triplet M. leonina, M. mulatta, and M. thibetana, we inferred that only 1.33% of the loci across the entire genome were introgressed. Moreover, we found that a mere 0.29% of the genetic loci supported discordant topologies and were introgressed, indicating that interspecific introgression was limited among the species examined (Fig. 2f; Supplementary Table S7). Therefore, QuIBL analysis suggests that ILS, rather than introgression, is the predominant factor underlying the phylogenetic discordance among the species.

Fig. 2.

Fig. 2.

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High levels of ILS and footprints of introgression contribute to gene tree heterogeneity. a, b) DensiTree plots based on 200-kb window trees spanning the whole genome. The top 2 most common topologies are represented by left a) and right b), respectively. The main incongruence between both trees is highlighted by the dashed boxes. c, d) The top 2 most common topologies were selected as reference trees and a heatmap is shown to illustrate the statistical support for introgression between species pairs inferred from Dsuit software. The f-branch function was used to further process the Dtrios results for every branch on the tree. Arrows indicate gene flow from either the ancestral silenus/nigra c) or sinica/arctoides d) clades into the ancestral fascicularis/mulatta clade. e) The diagram illustrates the complex evolutionary relationship between the macaque species, highlighting instances of hybridization and ILS. The fascicularis/mulatta clade is of hybrid origin, with the blue (silenus/nigra) and red (sinica/arctoides) γ corresponding to the estimated probabilities of inheritance from the respective ancestral clades. f) QuIBL results for 3 major Asian macaque clades. Relationships between the internal branch length in coalescent units (C2) and the total proportion of introgressed loci. Blue symbols represent triplets with the first most common topologies, and orange symbols represent triplets with the second most common topologies. The total proportion of introgressed loci was obtained by multiplying the probability that each topology corresponds to an introgression based on its genomic frequency. The figure shows that a large number of the most common topologies (i.e. the topology in a) have high non-ILS proportions, whereas the second most common topologies (i.e. the topology in b) has low non-ILS proportions when the internal branch lengths in coalescent units (C2) are short, which implies that the second most common topologies are primarily caused by ILS.

Contribution of Hybridization to Gene Tree Heterogeneity

Previous studies have suggested a vital role of hybridization in shaping the evolutionary history of macaques (Ciani et al. 1989; Evans et al. 2001, 2003; Tosi, Disotell, et al. 2003; Hamada et al. 2006; Li, Handsaker, et al. 2009; Li, Han, et al. 2009; Yan et al. 2011; Hamada et al. 2012; Fan et al. 2014, 2017; Fan et al. 2018; Ito et al. 2020; Song et al. 2021; Zhang et al. 2023). In the present study, we inferred footprints of ancient introgression across macaques using an approach based on D-statistics (Durand et al. 2011) implemented in Dsuite (Malinsky et al. 2021) (Fig. 2a and b; Supplementary Table S8 to S9). This method estimates D-statistics for all possible combinations of trios in macaques based on the 2 alternative topologies shown in Fig. 2a and b and then performs an f-branch test to assign gene flow to specific internal branches (Fig. 2c and d). The f-branch test based on the species tree (Fig. 2a) suggested an introgression event between the ancestral fascicularis/mulatta and silenus/nigra lineages (Fig. 2c), whereas for the second most common topology (Fig. 2b), introgression between ancestral fascicularis/mulatta and arctoides/sinica lineages was evident (Fig. 2d). Next, we determined the number of reticulation events using the maximum pseudo-likelihood (MPL) algorithm in PhyloNet to study ancient hybrid speciation (Yu et al. 2014). This algorithm identifies reticulated nodes for the hybridization scenario between the ancestors of the fascicularis/mulatta, arctoides/sinica, and silenus/nigra lineages (Supplementary Fig. S9). Specifically, we observed an inheritance probability of approximately 30% between the silenus/nigra lineage and the fascicularis/mulatta lineage, which is consistent with the distribution of quartet scores and phylogenetic signals obtained from GF (log-likelihood scores = −10,786).

Next, we corroborated the evidence for the hybrid origin of the fascicularis/mulatta clade using hybridization detection (HyDe) (Blischak et al. 2018), which examines genome-scale data for a large number of taxa and identifies the population that may have arisen through hybrid speciation as well as its putative parental populations, by estimating an inheritance parameter (γ value)⁠ to quantify the genomic contribution of the parents to the hybrid. In the present study, we used HyDe to determine the significance level of hybridization for all clade combinations, including P. anubis as an outgroup and triplets of macaque in-group taxa. However, only 1 of the 4 significant hybridization events yielded a γ value of 0.55 (Z-score = 306.84; P = 0) (Supplementary Table S10), which indicates that the fascicularis/mulatta lineage is a hybrid product with genomic contributions of about 55% and 45% from the arctoides/sinica and silenus/nigra lineages, respectively (Supplementary Table S11). Next, we verified hybridization for the fascicularis/mulatta lineage at the individual level and found that all 5 individuals of this clade exhibited γ values of 0.534 to 0.571, further supporting the hybrid origin of the fascicularis/mulatta lineage (Supplementary Table S11). Finally, bootstrap resampling of individuals within hybrid populations was performed to obtain a distribution of gamma values to assess heterogeneity for the levels of introgression (Fig. 2e).

Finally, we used Fd statistics (Martin et al. 2015) to calculate potential introgressed signals within 10-kb windows and considered the top 5% of the genomic regions with the highest Fd values as potential introgressed regions. Given the challenge to accurately identify introgressed regions in macaque genomes, we applied a 2-layer hidden Markov model (Guan 2014) to infer the local ancestry of the admixed individuals of the fascicularis/mulatta lineage. We selected the top 1% of the windows with a high sum of the proportion of source population (silenus/nigra lineage) ancestry for all SNVs in 10-kb windows with high Fd values as potential introgression regions (Supplementary Table S12) and identified some functional genes (e.g. CERS3, ACER3, and GSK3B) in these regions (Supplementary Fig. S10 and Table S13). Next, we reconstructed a phylogenetic tree based on autosomal SNVs including only the SNVs located in all putatively introgressed regions of the fascicularis/mulatta lineage that were derived from the silenus/nigra lineage. We observed that these lineages clustered together with the arctoides/sinica lineages to form their sister clade (Supplementary Fig. S11). We then reconstructed a phylogenetic tree excluding SNVs in these putatively introgressed regions and used only those in likely nonintrogressed regions to obtain a tree topology (Supplementary Fig. S11), which was identical to the species tree (Fig. 1b). Accordingly, trees based on introgressed and nonintrogressed regions resulted in different phylogenetic positions for the fascicularis/mulatta lineage.

Divergence among Macaques

Based on our divergence time estimations, the split between Macaca and Papio occurred in the Late Miocene period, approximately 7.08 Ma (95% highest posterior density [HPD] 8.21 to 5.88) (Fig. 3; Supplementary Fig. S12). Among the macaques, M. sylvanus diverged from Asian macaques at 4.72 Ma (95% HPD 5.47 to 3.92). In Asia, the divergence of species groups occurred in the Pliocene era, beginning with the split between the silenus/nigra clade and the other 4 species groups at 3.69 Ma (95% HPD 4.28 to 3.06), followed shortly afterward by the separation of the ancestors of the fascicularis/mulatta and arctoides/sinica clades (3.53 Ma, 95% HPD 4.1 to 2.93). These 3 clades finally diverged into species groups at 3 Ma (95% HPD 3.48 to 2.49; silenus and nigra groups), 2.82 Ma (95% HPD 3.27 to 2.34; sinica and arctoides groups), and 2.74 Ma (95% HPD 3.18 to 2.27; mulatta and fascicularis groups). Speciation within species groups occurred in the Pleistocene era at 2.46 to 1.31 Ma.

Fig. 3.

Fig. 3.

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Phylogenetic tree showing divergence times among macaques that were estimated from 3,319,411 4D sites from 10,641 single-copy genes. Bars indicate 95% confidence intervals of divergence times and the time scale below indicates Ma. Four fossil-based calibration points are as follows: Catarrhini 20 to 38 Ma, Hominidae 13 to 18 Ma, Homo-Pan 6 to 7 Ma, and Papio-Macaca 5.33 to 12.51 Ma. E, early; L, late; M, middle.

Demographic History

Changes in the effective population size (Ne) of the macaque species over time were modeled based on the distribution of heterozygous sites across the genome using a pairwise sequentially Markovian coalescent (PSMC) model (Li and Durbin 2011) (Supplementary Figs. S13a and d and S14). With the exception of M. sylvanus (Supplementary Fig. S13d), which exhibited a truncated plot most likely resulting from insufficient sequencing depth, the Ne of the remaining macaque species was very similar from 5 Ma until ∼2.5 Ma. This indicates that all Asian macaque species shared the same demographic history during the Pliocene era. The Pliocene was a period of global cooling after the warmer Miocene era and the cooling and drying of the global environment may have contributed to the enormous spread of savannas during this time (Salzmann et al. 2011). The change in vegetation undoubtedly has a significant impact on arboreal animals, such as macaques. Subsequently, the ancestor of most macaque species exhibited an upward trend of Ne after ∼2.5 Ma. However, the Ne of 3 nigra group species remained stable until 1.5 Ma, with a subsequent upward trend at different rates, possibly corresponding to population growth and expansion once favorable ecological conditions were present. Macaques, except of the nigra group, experienced a population growth until the Pleistocene glaciations and the Ne of different macaque species declined during different glaciations, indicating that they were likely restricted to smaller refugia during glacial periods. During the last interglacial period, Ne for M. mulatta (CR), M. assamensis, M. nemestrina, and M. tonkeana increased rapidly but subsequently declined again.

Discussion

Phylogeny and Classification of Species Groups

In the present study, we verified the validity of species groups within the Macaca genus and reconstructed their evolutionary history, which represents an exciting evolutionary scenario as a primate taxon, whose spread “Out of Africa” mirrors that of non-African humans. The species tree generated by ASTRAL supports the division of macaques into 7 well-supported clades or lineages that correspond to the classification of the genus into 7 species groups (Zinner et al. 2013; Roos et al. 2014) (Fig. 1b). M. sylvanus diverged first and the FST between M. sylvanus and Asian macaques was higher than among Asian macaque groups (Supplementary Table S5). These results support the separation of the sylvanus from the silenus group, thus confirming the classification of Delson (1980) based on morphology and geographic distribution. Asian macaques consist of silenus and nigra lineages and a clade including all other Asian lineages (Fig. 1b). The silenus and nigra lineages were grouped into a single species group (silenus group) by Fooden (1976) and Delson (1980) but were recently separated into 2 distinct species groups: silenus and nigra or Sulawesi (Groves 2001; Zinner et al. 2013; Roos et al. 2014, Roos et al. 2019). Based on the species tree, the 6 species of the silenus and nigra groups that we examined cluster into 2 clades: silenus group (M. silenus, M. leonina, and M. nemestrina) and nigra group (M. nigra, M. maura, and M. tonkeana) (Fig. 1b).

The other Asian macaques may be divided into 2 major clades. The first one includes M. fascicularis, M. fuscata, M. cyclopis, and M. mulatta (Fig. 1b). Initially, these 4 species were grouped into the fascicularis group (Fooden 1976); however, Groves (2001) separated M. fuscata, M. cyclopis, and M. mulatta from the fascicularis group into the own species group, mulatta group. The FST values varied widely between M. fascicularis versus M. mulatta (0.171) and M. fascicularis versus M. fuscata (0.415). Yan et al. (2011) indicated that approximately 30% of the genomes of mainland M. fascicularis are of Chinese M. mulatta origin, which explains the lower genetic distance between these 2 species in our analysis. However, M. fascicularis from the Sundaland region may not contain any genomic contribution of M. mulatta, which likely results in increased FST values, thus justifying to separate them into distinct species groups. The second major clade consists of M. arctoides, M. assamensis, M. thibetana, M. leucogenys, and M. sinica (Fig. 1b). M. arctoides was recently assigned to its own species group, the arctoides group (Zinner et al. 2013; Roos et al. 2014, 2019), but due to morphological similarities traditionally classified as a member of the sinica group (Delson 1980; Tosi et al. 2000; Li, Handsaker, et al. 2009; Li, Han, et al. 2009), while Groves (2001) recognized M. arctoides as a member of the fascicularis group. M. arctoides should be separated from the sinica group in its own species group.

Contributions of ILS and Ancient Hybridization to Gene Tree Heterogeneity

In a recent study, gene flow between the fascicularis/mulatta and silenus/nigra groups was reported. It was concluded that the introgression signals between both groups are primarily the result of genome similarity in closely related species (Song et al. 2021). In the present study, we used QuIBL to show that among 120 triplets, 60.8% exhibited phylogenetic discordances exclusively resulting from ILS. Moreover, evidence for introgression was only observed in 9.1% of the triplets (Supplementary Table S7), indicating that ILS, rather than introgression, is the primary factor that contributes to the observed gene tree discordance. However, our results also suggest that ancient hybrid speciation may have occurred among the ancestral populations of the silenus/nigra and arctoides/sinica clades. This resulted in the formation of the fascicularis/mulatta clade, although these analyses do not differentiate between ILS and introgression, which can both create similar phylogenetic signals (Fontaine et al. 2015). Thus, a plausible explanation is that the fascicularis/mulatta clade may have originated from an ancient hybrid speciation of 2 parental lineages (the ancestral populations silenus/nigra and arctoides/sinica clades), which was undoubtedly accompanied by a high level of ILS. The reason why we are confident in the notable incidence of ILS is that, according to PSMC analysis, all species in the genus Macaca experienced a population decline during the time window of the potential hybridization event (3.53 to 3.69 Ma). Population declines in ancestral lineages result in rapid segregation into small isolated subgroups resulting in a rapidly diverging lineage. Furthermore, the substantial fraction of gene trees that are incongruent with the species tree in the genome, also referred to as hemiplasy (Avise and Robinson 2008), is particularly prevalent when ancestral populations are large and the time interval between speciation events is short. Hemiplasy may be attributed to the stochastic segregation of ancestral allelic polymorphisms from ancestrally large effective populations into already diverging subpopulations within a relatively short time interval (Whitfield and Lockhart 2007; Steenwyk et al. 2023). Thus, all of this constitutes a prerequisite for high levels of ILS at the ancestral nodes of the Asian macaque radiation. With respect to potential hybridization events, our Dsuit results based on the 2 most common tree topologies (Fig. 2c and d), PhyloNet (Supplementary Fig. S10) and HyDe (Fig. 2e; Supplementary Tables S10 and S11), suggest the fascicularis/mulatta clade as a potential hybrid product. However, the authenticity of this event remains under discussion because of 2 reasons. First, it is difficult to accurately detect the superposition of multiple introgression events throughout the evolutionary history of macaques, and ancient hybridization events may have subsequent unique evolutionary histories, such as ongoing gene flow, distinct introgression histories, maintenance of assortative mating, and rate heterogeneity (Sankararaman et al. 2014). Second, the power of HyDe and D-statistics to detect hybridization events in the presence of high rates of ILS and ancient rapid radiation events is limited (Kong and Kubatko 2021). Simulation analyses revealed that in the case of high levels of ILS, the power of HyDe decreases to 0 to 0.1, and the power of D-statistics was relatively poor (0.03 to 0.7) either. Furthermore, the accuracy of HyDe for the γ estimates of hybridization events relative to ancestry is further reduced and accompanied by a high false-positive rate (Kong and Kubatko 2021). The reason therefore is that ILS contributes to both ABBA and BABA counts, thereby diluting information and masking the signature of hybridization in the hybrid genome. Individuals or taxa with longer external branch lengths have more time to accumulate mutations, and the specific pattern of loci that are informative for detecting hybridization may be lost (Kong and Kubatko 2021). Although the authenticity of hybridization cannot be confirmed in the present study, we believe that both the high levels of ILS and potential hybridization contributed to the genealogical discordance observed across the genome. Our study and that of others show that macaque individuals, even from the same species and population, can exhibit quite distinct genetic backgrounds (Xue et al. 2016; Liu et al. 2018). For a better understanding of macaque evolution and the extent of hybridization, more individuals should be evaluated in future studies.

The Radiation of Macaques

Fossil data suggest that the genus Macaca arose about 7 Ma in Northern Africa during the Late Miocene era (Delson 1980; Whitfield and Lockhart 2007; Salzmann et al. 2011; Fontaine et al. 2015), which is consistent with our MCMCTree results (7.08 Ma; Fig. 3). The Messinian salinity crisis (5.9 to 5.3 Ma), in which the level of the Mediterranean Sea fluctuated and occasionally dried out, may have facilitated macaque dispersal from North Africa (Hamada et al. 2006; Elton and O'Regan 2014). A large number of macaque fossils have been discovered in Europe near the Mediterranean (Hamada et al. 2006; Roos et al. 2019). Macaque ancestors likely invaded Europe via the arid Mediterranean during Messinian stage (Tosi, Disotell, et al. 2003; Tosi, Morales, and Melnick 2003; Hamada et al. 2006; Roos et al. 2019). The M. sylvanus ancestor split from the main stem at ∼4.72 Ma and settled around the Mediterranean (Fig. 3; Supplementary Fig. S15). Dental specimens from the oldest known Asian fossil, referred to as the genus Macaca, are Macaca palaeindicus from the Late Pliocene Tatrot formation of the Siwalik Hills (Delson 1980; Salzmann et al. 2011; Sankararaman et al. 2014). Hence, the ancestor of Asian macaques spread eastward step by step likely along the Siwalik Hills to the Hengduan Mountains, which are a potential diversification hotspot for primates (Peng et al. 1993; Jablonski 1998; Thinh et al. 2010; Roos et al. 2011), and diverged into different lineages. First, the ancestor of silenus/nigra split at ∼3.69 Ma and then potentially hybridized with the ancestor of other Asian macaques and formed a pattern of 3 clades (Supplementary Fig. S15).

Conclusion

We conducted a phylogenetic analysis of 24 whole macaque genomes, of which 16 were newly generated. We identified ∼20 million high-quality SNVs for each macaque individual, which can be used as important baseline data for future studies. We found support for the division of macaques into 7 species groups and observed visible differences in FST within and between species groups. We also conclude that ILS is the main factor that caused gene tree discordances in macaques, rather than introgression. Specifically, the conflicting phylogenetic position of the fascicularis/mulatta is likely the result of high levels of ILS and potential hybridization between ancestors of the arctoides/sinica and silenus/nigra lineages. In addition, we used the available fossil data along with the literature to reconstruct the radiation scenario of the macaques.

Materials and Methods

Sample Collection and Library Preparation

Samples of 16 macaque individuals were obtained from the Institute of Zoology, Chinese Academy of Sciences and the German Primate Center (Supplementary Table S1). Genome data for 8 other macaque individuals and 1 P. anubis individual were obtained from the NCBI Short Read Archive (for accession number, see Supplementary Table S1). The present study was approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. The procedure for blood and tissue collection was in strict accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China. DNA was isolated from the blood or tissue samples using the Qiagen Blood and Tissue Kit. Sequencing libraries were prepared based on the KAPA library preparation kit with insert sizes of 300 to 500 bp and sequenced using Illumina HiSeq 2500 and 4000 technologies. Quality control was performed using FastQC with default parameters (www.bioinformatics.babraham.ac.uk/projects/fastqc/).

Data Processing

Paired-end reads for all samples were mapped to the P. anubis reference genome (Panu_3.0, GCA_000264685.2) using the Burrows–Wheeler Aligner mem v0.7.17 (Li and Durbin 2009), except for M. nemestrina (2), which was trimmed with cutadapt v2.4 (Kechin et al. 2017) first, because of poor sequencing quality resulting from DNA degradation. The output bam files were sorted using SAMtools (Li, Handsaker, et al. 2009; Li, Han, et al. 2009), and duplicates were marked with GATK v4.1.2.0 (McKenna et al. 2010). SNVs were called using GATK following best practice. We obtained a GVCF file for each individual using the “HaplotypeCaller” method in GATK, and the samples were merged based on “CombineGVCFs.” We used the GenotypeGVCFs-based method with the “includeNonVariantSites” flag to obtain the vcf files. Next, we applied “SelectVariants” to exclude indels and split the variant and nonvariant sites. All nonvariant sites were filtered in subsequent analyses. We applied the hard filter command “VariantFiltration” to exclude potential false-positive variant calls using the following criteria: “-filterExpression QD < 5.0 || FS > 60.0 || MQ < 40.0 || ReadPosRankSum < −8.0 || MQRankSum < −12.5” and “-genotypeFilterExpression DP < 4.0” (Van der Auwera et al. 2013). All variant calls failing the filter were removed. All SNVs were annotated using ANNOVAR v2013–06-21 (Wang et al. 2010) (Supplementary Table S3) based on the gff3 file from Ensembl (Papio_anubis.Panu_3.0.104.gff3). Heterozygosity was defined as the rate of heterozygous SNVs in the genome, which were further filtered using the following parameters: “--minGQ 20” and “--minDP 8” to ensure data quality based on VCFtools. We calculated the heterozygosity rate in nonoverlapping windows of 100 kb in size for all 24 macaques. The genome-wide distribution of heterozygosity is shown in a box plot using boxplot from the R package (Supplementary Table S2 and Fig. S2).

Phylogenetic Reconstruction Using a Window-Based Strategy

We constructed consensus sequences for all individual genomes and aligned them per chromosome (sex chromosomes, mitogenomes, and scaffolds were excluded) based on VCF, which did not include variants present in the Papio reference, but were invariant among macaques. In addition, indels, repetitive regions, and low-quality regions were removed from the consensus sequences. Per-chromosome alignments were split into nonoverlapping windows of 200 kb. For each window, we constructed ML trees with RAxML v8 (Stamatakis 2014) using the GTR + CAT substitution model and 100 bootstrap replicates. Using the 200-kb window trees, we reconstructed a species tree using ASTRAL v4.10.5 (Sayyari and Mirarab 2016) under the MSC model returning quartet scores and PPs. The species tree was rooted with P. anubis. Consensus networks of the window trees were generated using SplitsTree5 (Huson and Bryant 2006) using different thresholds. Genealogical discordance across the rhesus macaque genome was assessed by Twisst (Martin and Van Belleghem 2017). All GF trees were clustered using PhyBin (https://github.com/rrnewton/PhyBin), and the top 2 topologies were visualized by DensiTree (Bouckaert 2010).

Phylogenetic Analysis of Concatenated Whole-Genome SNV Data

SNVs were extracted from the filtered VCF file and converted into individual FASTA sequences using a python script (https://doi.org/10.5281/zenodo.2540861). The FASTA sequences were merged into a single file to provide a multiple sequence alignment for all individuals and all concatenated SNVs. An ML phylogenetic tree was reconstructed with RAxML v8 using the GTR + CAT substitution model and 100 bootstrap replicates. In addition, a NJ tree was constructed using the TreeBeST v1.9.2 program (http://treesoft.sourceforge.net/treebest.shtml), which has a built-in algorithm to generate the best tree that reconciles with the species tree and is rooted with the minimal number of duplications and losses. To further visualize genetic relationships among the macaques, we performed PCA on the filtered autosomal SNVs using the Eigensoft package v5.0 (Patterson et al. 2006).

Phylogeny of Mitochondrial Genomes

We used NOVOPlasty (Dierckxsens et al. 2017) to de novo assemble the mitochondrial genomes (mitogenomes) of 18 macaques representing 15 species (Supplementary Table S4) and annotated them with MitoZ (Meng et al. 2019). In addition, we downloaded 15 additional macaque mitogenomes (Supplementary Table S4) from the NCBI based on the filter criteria mentioned in Roos et al. (2019). The 33 mitogenomes were aligned using MAFFT v7 (Yamada et al. 2016) and indels and poorly aligned positions were removed with Gblocks v0.91b (Castresana 2000). An ML tree was reconstructed using RAxML v8 with a GTR + CAT substitution model and 1,000 bootstrap replicates.

QuIBL

QuIBL (Edelman et al. 2019) was used to determine the likelihood of introgression and ILS for each locus in all species triplets. QuIBL provides estimates of the proportion of introgression and the probability of a locus falling into a model with either only ILS or introgression and ILS. QuIBL estimates the internal branch length distribution for each locus in the species triplet. Given its sensitivity to recombination (Edelman et al. 2019), we extracted 20-kb windows separated by 400 kb to minimize the risk of including a recombination breakpoint in the window. We then filtered the inferred ML trees based on the number of parsimony-informative sites (≧10), which resulted in a total of 3,175 trees that served as QuIBL inputs. We used the species tree topology to assign an outgroup to each triplet and calculated the percentage of loci that supported discordant topologies and showed significant evidence of introgression.

To determine whether ILS (scenario 1) or a combination of ILS and introgression (scenario 2) contributed to the observed gene tree discordances, we calculated QuIBL delta BIC values for inner branch lengths. To determine the most suitable model, differences in delta BIC were calculated by subtracting the delta BIC value of scenario 1 from the delta BIC value of scenario 2. When the difference in the delta BIC score is greater than 10, scenario 1 (only ILS) is preferred. If less than −10, scenario 2 (ILS and introgression) is preferred. When the difference is in the range of −10 to 10, both scenarios are indistinguishable.

D-statistics

We implemented D-statistics with Dsuite (Malinsky et al. 2021) across all combinations of the 16 macaque species described above. The topology required for Dsuite was consistent with the 2 most common tree topologies described in Figs. 1c and 2a and b. The D-statistics for all possible combinations with P. anubis as the outgroup (Out) and 3 different macaque species with a phylogenetic topology following (((M1, M2), M3), Out) were calculated using the Dtrios module. A total of 566 groupings were analyzed for each topology. D-statistics were calculated for each branch of alternative topologies using the f-branch module, and the statistical results were visualized using the dtools.py script provided with the Dsuite software.

Detection of Hybridization

To obtain further support for ancient introgression as indicated by D-statistics, we applied HyDe (Blischak et al. 2018), which automates the detection of hybridization across large numbers of species, despite ILS, and can test a hypothesis at the population or individual level by estimating the amount of admixture (γ). The procedure is as follows: the population approach was used in the “run_hyde_mp.py” script to test hybridization events among all triplet combinations among the 4 macaque clades (fascicularis/mulatta clade, arctoides/sinica clade, silenus/nigra clade, and sylvanus clade) with P. anubis as the outgroup. The results were first filtered based on whether there was significant evidence of hybridization (P < 0.05). In addition, we estimated admixture levels using gamma (γ) statistics. Values of approximately 0.5 indicate a hybrid origin with a 50:50 contribution of both parental lineages, whereas extremely low or high values suggest the absence of hybridization events. We found that values of approximately 0.5 were significantly concentrated among the silenus/nigra, fascicularis/mulatta, and arctoides/sinica clades. Next, we used the script “run_hyde_mp.py” to test each individual within a putative hybrid population (fascicularis/mulatta clade) using specified triplets. We then performed a bootstrap resampling (500 replicates) of the individuals within the putative hybrid lineages for each specified triplet based on the “bootstrap_hyde.py” script.

ML Inference for Reticulation with PhyloNet

We used the InferNetwork_MPL program in PhyloNet (Yu et al. 2014), which is based on the MPL algorithm, to analyze a set of every 3rd GF ML tree. In other words, 2,150 trees in a coalescent framework that accounts for ILS, while allowing for different numbers of reticulation events. To reduce the complexity and computational demand, we used only 1 individual per species. We set the number of reticulation events at 1 and 2 to conduct 2 independent analyses and each was run with 100 iterations to yielding 5 networks with the highest likelihood scores. We visualized the optimal networks in Dendroscope v3 (Huson and Scornavacca 2012).

Fd Value

To identify putatively introgressed regions in macaques, we followed the method described by Martin et al. (2015) and van der Valk et al. (2020). The method predicts that introgression between species in a specific genomic region should reduce the between-species divergence in this region compared with the rest of the genome (Martin et al. 2015; van der Valk et al. 2020). We computed the Fd value, which is sensitive to the number of informative sites within windows, for each 10-kb window across the whole genome of each macaque.

Introgression Analysis with Efficient Local Ancestry Inference

The 2-layer admixture model implemented in efficient local ancestry inference (Guan 2014) was used to infer introgressed segments in fascicularis/mulatta (the admixed population, -p 1) with silenus/nigra as source population 2 (-p 11) and arctoides/sinica as source population 1 (-p 10). We ran 30 separate iterations, setting the mixture generation values to 2.91 million, 3.2 million, and 3.45 million (based on the results of MCMCTree), and used the average of 3 independent runs to identify introgression events. Sites with a proportion of >1.5 from either source population were defined as introgression sites. We then calculated the sum of proportions >1.5 from each 10-kb window for each source population and defined the top 1% of the windows as putative introgression tracts.

Analysis of Demographic History Using Genome-Wide Data

We performed a PSMC (v0.6.5-r67) analysis (Li and Durbin 2011) to reconstruct the demographic history of each macaque individual. To enhance mapping quality, we set parameter “-C” to 50 and created input files for PSMC using the SAMtools mpileup module (Li, Handsaker, et al. 2009; Li, Han, et al. 2009). Only autosomal SNVs were reserved to generate the diploid sequence for PSMC. PSMC was run with 25 iterations (-N), a maximum 2N0 coalescent time (-t) of 15, an initial theta/rho ratio (-r) of 5, and the 64 time intervals were parameterized as “4 + 25 ∗ 2 + 4 + 6.” PSMC plots were scaled with a mutation rate (μ) of 0.8 × 10−8 and a generation time (g) of 11 yr for all macaque species (Xue et al. 2016). Bootstrap analyses (100 bootstrap replications) were performed for each species.

Divergence Time Calibration

Coding sequences (CDSs) for different representative outgroup species and 3 macaques (M. mulatta, M. fascicularis, M. nemestrina, P. anubis, Homo sapiens, Pan troglodytes, and Pongo abelii) were retrieved from Ensembl (release-98). The list of single-copy orthologous genes shared by these species was obtained by OrthoFinder (Emms and Kelly 2019). The macaques were mapped to the Panu_3.0 genome; thus, their CDSs have the same genomic coordinates. Therefore, the CDSs of all macaques were extracted after ortholog detection. CDSs of these single-copy genes were aligned with PRANK v.170427 (Löytynoja 2014) using the codon model and concatenated into a single supermatrix. Fourfold degenerate (4D) sites were extracted using MEGA v7 (Kumar et al. 2016) and concatenated to estimate the divergence times with MCMCTree in PAML v4.9 (Yang 2007). To calibrate the molecular clock, we set constraints on 4 nodes: Catarrhini 20 to 38 Ma, Hominidae 13 to 18 Ma, Homo-Pan 6 to 7 Ma, and Papio-Macaca 5.33 to 12.51 Ma (Perelman et al. 2011; Roos et al. 2019; de Vries and Beck. 2023). To check for convergence of the stationary distribution, the analysis was run in duplicate and the results of both runs were compared.

Supplementary Material

msad229_Supplementary_Data
Click here for additional data file. (5.2MB, zip)

Acknowledgments

The authors thank Prof. Luo Shujin and Dr. Sun Xin for their assistance in DNA isolation, library preparation, and quality control for M. nemestrina (2).

Contributor Information

Xinxin Tan, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Geneplus-Beijing Institute, Beijing 102206, China.

Jiwei Qi, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Zhijin Liu, College of Life Sciences, Capital Normal University, Beijing 100049, China.

Pengfei Fan, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China.

Gaoming Liu, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Liye Zhang, Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göttingen 37077, Germany.

Ying Shen, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Jing Li, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China.

Christian Roos, Primate Genetics Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göttingen 37077, Germany; Gene Bank of Primates, German Primate Center, Leibniz Institute for Primate Research, Göttingen 37077, Germany.

Xuming Zhou, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Ming Li, CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Supplementary Material

Supplementary material is available at Molecular Biology and Evolution online.

Author Contributions

M.L. conceived this study, and X.Z. and C.R. contributed to the idea for this study. X.T., J.Q., and Z.L. performed the experiments, analyzed the data, and wrote the draft manuscript. G.L., L.Z., and Y. S. helped to analyze data. P.F., C.R., and J.L. provided samples. M.L., X.M., and C.R. revised the draft manuscript. All authors contributed to the final text.

Funding

The work was supported by the National Natural Science Foundation of China (31821001 and 31530068), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB31000000 and XDA23080000), and Sino-German Mobility Programme (M-0084).

Data Availability

Whole-genome resequencing data produced in this study were deposited at the NCBI Sequence Read Archive (SRA) under Bioproject PRJNA732172.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

msad229_Supplementary_Data
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Data Availability Statement

Whole-genome resequencing data produced in this study were deposited at the NCBI Sequence Read Archive (SRA) under Bioproject PRJNA732172.

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