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Sex-biased migration and admixture in macaque species revealed by comparison between autosomal and X-chromosomal genomic sequences

The role of sex-specific demography in hybridization and admixture of genetically diverged species and populations is essential to understand the mechanisms forming the genomic diversity of sexually reproducing organisms. In order to infer how sex-linked genetic loci have been differentiated undergoing frequent hybridization and admixture, we examined 17 whole-genome sequences of seven species of the genus Macaca, which shows frequent inter-specific hybridization and predominantly female philopatry. We found that hybridization and admixture were prevalent within these species. For three cases of suggested hybrid origin of species/subspecies, M. arctoides, M. fascicularis ssp. aurea, and Chinese M. mulatta, we examined the level of admixture of X chromosomes, which is less affected by male-biased migration than that of autosomes. In one case, we were able to determine that M. cyclopis and M. fuscata was genetically closer to Chinese M. mulatta than to the Indian M. mulatta, and the admixture level of Chinese M. mulatta and M. fuscata/cyclopis was more pronounced on the X chromosome than on autosomes. Since the mitochondrial genomes of Chinese M. mulatta, M. cyclopis, and M. fuscata were found to cluster together, and the mitochondrial genome of Indian M. mulatta is more distantly related, the observed pattern of genetic differentiation on X-chromosomal loci is consistent with the nuclear swamping hypothesis, in which strong, continuous male-biased introgression from the ancestral Chinese M. mulatta population to a population related to M. fuscata and M. cyclopis generated incongruencies between the genealogies of the mitochondrial and autosomal genomes.


Introduction
A key issue in evolutionary genetics is understanding the mechanisms by which populations are genetically differentiated and continue diverging to form separate species. Genetic differentiation is often attributed to geographical isolation, and genetically differentiated populations evolve into new, reproductively isolated, species. However, hybridization and admixture among genetically divergent populations occasionally occur, impeding further genetic differentiation. Hybridization and admixture have frequently been observed in populations of wild animals and between different species ( Green et al. 2010;Osada et al. 2010;Meyer et al. 2012;Cahill et al. 2015;Fan et al. 2018). S i m i l a r l y , t he migration of males is expected to have less impact on X chromosomes than on autosomes, because a female carries two X chromosomes but a male has only one. Genetic differentiation at these sex-linked markers, therefore, provides insights into the way in which sex-biased migration has shaped animal genomes through hybridization and admixture among populations and species ( Heyer and Segurel 2010).
In order to investigate patterns of genetic differentiation across genomes during hybridization process, and to estimate how sex-biased migration contributes to species divergence, we focused on macaque monkeys. The genus Macaca consists of 24 species distributed in the part of the Eurasian and African continents, mostly in the South, Southeast, and East Asia Roos et al. 2014). They are classified into four to seven species groups, depending on the criteria used Roos et al. 2019). In this study, we followed the grouping proposed by , and focused on four out of seven species groups: the fascicularis group, including Macaca fascicularis showed that the fascicularis and mulatta groups and the sinica and arctoides groups are sister pairs ( Fan et al. 2018).
Previous studies have revealed that ancient inter-specific gene flow has been common in macaques, not only between sister species such as M. fascicularis and M. mulatta ( Osada et al. 2010;Yan et al. 2011) but also between different species groups, for example, M. thibetana in the sinica group and M. mulatta in the mulatta group ( Fan et al. 2014). Ancient hybridization between the arctoides and mulatta groups was also supported by whole-genome sequencing analysis of M. arctoides ( Fan et al. 2018). The divergence of the sinica-arctoides and fascicularis-mulatta groups is estimated to have occurred around 1.5-2.0 Mya, as per fossil evidence ( Delson 1980), but much earlier estimates ( 2.8-4.9 Mya) have been obtained using nucleotide sequences ( Perelman et al. 2011;Jiang et al. 2016;Matsudaira et al. 2018;Roos et al. 2019).
In some macaque species, discordant genealogies have been obtained using autosomal, mitochondrial, and Y-chromosomal loci, a situation potentially reflecting sex-biased migration patterns in the past ( Tosi, Morales, and Melnick 2000;Tosi, Morales, and Melnick 2002;Tosi, Morales, and Melnick 2003;Evans et al. 2010;Zinner, Arnold, and Roos 2011 ( 2) M. fascicularis ssp. aurea has been found to be distributed in Myanmar and Thailand, having a morphology distinct from the other nine subspecies (Fooden, 1995). A genetic study using mitochondrial and Y-chromosomal sequences showed that M. fascicularis ssp. aurea clustered with the sinica group at the mitochondrial locus, but clustered with the fascicularis group at the Y-chromosomal locus ( Matsudaira et al. 2018).  ( Zinner, Arnold, and Roos 2011;. This is referred to as the nuclear swamping hypothesis. A schematic representation of the nuclear swamping hypothesis is presented in Figure 1. However, in many species other than primates, introgression of mitochondrial genomes between species, so-called mitochondrial capture, is not rare ( Bachtrog et al. 2006;Veale, Russell, and King 2018). Since the effective population size of the mitochondrial genome is much smaller than that of autosomal genomes, introgressed mitochondrial genomes might easily become fixed in the recipient population by genetic drift ( Osada 2011). Besides, in several animal species, paternal leakage of mitochondrial genomes has been observed, particularly in cases of inter-specific hybridization ( Rokas, Ladoukakis, and Zouros 2003;Mastrantonio, Urbanelli, and Porretta 2019). Therefore, in order to determine the factors underlying incongruent genealogies between autosomal and sex-linked loci, a study focusing on sex-biased migration using loci other than mitochondrial locus is desirable. Genetic differentiation at the X chromosome has the potential to provide a picture with substantially higher resolution in the estimation of sex-biased migration rates, because X chromosomes are longer than mitochondria and, more importantly, recombine except at the pseudo-autosomal regions In order to investigate how sex-biased migration affects the inter-specific hybridization of genomes, we used the above-mentioned three cases that showed incongruent species trees between mitochondrial and nuclear loci: ( 1) between M. arctoides and the mulatta-group species; ( 2) between M. fascicularis ssp. aurea and the sinica-group species; and ( 3) among the mulatta-group species. We clarified the phylogeny of eight macaque species using 17 whole-genome sequences, by combining newly and previously determined whole-genome sequences of macaques, and compared the level of genetic admixture at the mitochondrial genome, autosomes, and X chromosomes.

Whole-genome sequencing and genetic diversity in populations
This study has performed whole-genome sequencing of five macaque samples: one female M.  Table 1). We also obtained short-read sequences of an additional 12 samples from previous studies

Species phylogeny
To examine the phylogenetic relationships among the macaques, we first constructed a tree using mitochondrial genomes Next, we constructed a neighbor-joining tree using the genetic distance between nuclear genomes with the draft genome sequence of OLB as an outgroup any priori assumptions about their population structure. The f3 statistics showed that CMA1 and CMA2 were generally closer to CMT and CMV than to CMM or the populations in the mulatta group ( Figure 4A). For example, the outgroup f3 of CMA1 and CMT was significantly greater than that of CMA1 and RMC The phylogenetic relationship among the three species of the mulatta group-M. mulatta, M.
fuscata, and M. cyclopis-has not previously been examined at the whole-genome level. In the neighbor-joining tree shown in Figure 3, the Chinese M. mulatta samples clustered with the Indian M. mulatta samples, and M. mulatta was monophyletic and sister to M. cyclopis.
However, the branching pattern of the tree may be distorted by gene flow between species/populations. The f3 statistics agreed with the pattern in the neighbor-joining tree and indicated that M. mulatta and M. cyclopis were the sister pair ( Figure 4B).

General picture of admixture between species/populations
We have examined the presence of gene flow between species/populations using f4 statistics.
Here we represent the configuration of f4 statistics as f4(A, B; C, D), where A-D represent the names of the populations to which the samples belong. We fixed the species A to OLB, since we assumed that OLB has not experienced any recent gene flow with our macaque populations. As   CMT were more closely related to RMC than to RMI.

Regional heterogeneity of genetic differentiation on the X chromosomes
Before estimating the level of admixture on the X chromosome, we investigated the regional heterogeneity of genetic differentiation on the X chromosome. We computed f4 statistics for each SNV across the non-pseudo-autosomal region of the X chromosome. We found that an approximately 11 Mb length region proximal to the pseudo-autosomal region 1 The f4 A values of the mulatta group were generally lower than those of the fascicularis group, indicating a strong affinity of STM to the mulatta group.
If the mulatta-like mitochondrial genomes of STM originated via nuclear swamping, we could expect that the X chromosomes of STM would be more closely related to those of the mulatta group and that f4 X showed that the f4 X had very large variance across the chromosome and were not significantly In addition, there was no consistent trend in the differences between the f4 X and f4 A, as shown in Figure 5A.
We next compared f4 A and f4 X including CMA1 and CMA2. We computed the f4(OLB, ASM; CMA1/2, D), where D represents a population in the fascicularis or mulatta group other than CMA1 and CMA2. The f4 A values were observed to be strongly negative, indicating that ASM was significantly more closely related to CMA1 and CMA2 than to the other fascicularisand mulatta-group populations ( Figure 5B), which coincides with the observation that the mitochondrial genomes of CMA1 and CMA2 clustered with those of the sinica group ( Figure   2).
Similar to the previous analysis, if the sinica-like mitochondrial genomes of CMA1 and CMA2 were the consequence of nuclear swamping, we would expect that the X chromosomes of CMA1 and CMA2 would be more closely related to those of the sinica group than to the autosomes, resulting in f4 X being lower than f4 A .
In one comparison, f4 X ( OLB, ASM; CMA1, CMV) was significantly lower than f4 A ( OLB, ASM; CMA1, CMV), supporting the nuclear swamping hypothesis; in the other comparisons, however, the f4 X values were not significantly different from or were significantly higher than those on autosomes. Again, the results did not consistently support the nuclear swamping hypothesis.
We finally looked at the f4 statistics to detect admixture among the species/populations in the mulatta group. We computed f4 A and f4 X values for f4(OLB, JPM; RMC, RMI) and f4(OLB, TWM; RMC, RMI). In both cases, the f4 A and f4 X values were significantly negative, indicating that JPM and TWM were more closely related to RMC than to RMI . We also found that f4 X was significantly lower than f4 A ( p = 0.0044 for JPM and p = 0.0008 for TWM, t-test), which shows that RMC were more similar to JPM and TWM on the X chromosomes than on autosomes, a pattern expected under the nuclear swamping hypothesis To support these observations, we constructed an admixture graph using autosomal data. The admixture graph suggested a complex admixture history of the mulatta group ( Figure 6).
Although the reconstructed admixture graph is only one of the good past demographic models that explains the data, the admixture graph was concordant with the hypothesis of two-round hybridization for the origin of Chinese M. mulatta inferred by the f4 statistics. Based on this model, the populations that diverged from the lineages of M. fuscata and M. cyclopis generated a hybrid population ( node K in Fig. 6), and later, this hybrid population admixed with the ancestral population of Chinese M. mulatta ( node O in Fig. 6).
We assume that the admixture graph inferred from the autosomal data reflects the past demographic events among species reasonably accurately and estimated the admixed fraction on the X chromosome by fixing the admixture graph ( Figure 6). In general, the level of admixture on the X chromosomes was lower than that on autosomes. However, the admixed fraction from the ghost population ( node L in Fig. 6) in the RMC genomes on the X chromosomes was 67 % , which was much higher than that on autosomes

Discussion
In this study, we examined 17 macaque genomes to investigate their evolutionary history.
Comparing the pattern of genetic differentiation between the autosomal and X-chromosomal genomes is the primary focus of this study, in order to quantify the effect of sex-biased migration, which is universally observed in sexually reproducing organisms. The number of samples per species in this study was limited, and we mostly neglected the genetic structure within species. In particular, M. mulatta and M. fascicularis are widely distributed, and they showed detectable levels of genetic structure within the species We should also note that the different patterns of genetic differentiation between autosomal and X-chromosomal loci expected under nuclear swamping are also explained by strong natural selection against the introgression of X chromosomes. These factors are not mutually exclusive, and their relative importance should be examined using multiple sources of evidence. In the case of Chinese M. mulatta, if we assume that the mitochondrial locus is evolutionarily neutral, 13 the pattern observed provides evidence supporting the nuclear swamping hypothesis. However, several studies have suggested that the introgression of mitochondrial genomes might be deleterious because they may disrupt the interactions between nuclear and mitochondrial alleles, a phenomenon which is referred to as mitonuclear incompatibility

Conclusions
In this study, we investigated the complex evolutionary history of macaques with rampant gene flow among species, using a whole-genome sequence dataset. As shown in previous studies, incongruencies in genealogical relationships between nuclear and mitochondrial genomes were observed among species. We showed that comparing the pattern of admixture between autosomal and X-chromosomal loci is a potentially valuable approach to statistically evaluate the power of sex-biased migration in shaping the pattern of genome evolution. We detected a statistically significant difference in admixture levels between these loci, which could be explained by strong male-biased migration in one of the three cases tested. Comparisons between an autosomal and an X-chromosomal evolutionary pattern using a larger dataset in other species would reveal a more detailed evolutionary history in future studies.

Variant calling
For all samples, reads were mapped to the reference genome sequence of OLB The heterozygosity of the samples was calculated using an in-house-generated Python script, which calls the number of homozygous and heterozygous nucleotide sites with coverage between 10 and 60. The segments of the ROH were estimated using the PLINK software ( Purcell et al. 2007).

Phylogenetic tree of mitochondrial genomes
We assembled mitochondrial genomes from short-read sequences using the NOVOPlasty software ( Dierckxsens, Mardulyn, and Smits 2016). Of the 17 samples we analyzed, 9 yielded circular mitochondrial genome assemblies. The five newly determined mitochondrial genome sequences were deposited in the public databases ( DDBJ/EMBL/NCBI accession numbers: LC536644-LC536648). The mitochondrial genomes of CMV1 was assembled using the same method with the data obtained by Yan et al.

Analysis of species phylogeny and admixture
To construct a neighbor-joining tree and neighbor-net network, all pairwise genomic distances were calculated. The genomic distance D between two diploid genomes, X and Y, was calculated using the following equation.
where a i b i and c i d i represent the genotypes of X and Y at the i-th site and δ pq indicates Kronecker delta. The neighbor-net network was constructed using the phangorn package in R    Schematic representation of the nuclear swamping hypothesis. The letters A, X, Y, and mt represent autosomal, X-chromosomal, Y-chromosomal, and mitochondrial genomes, respectively, and the circles indicate the gene pool of a population. When strong male-biased migration continues for a long time, the nuclear genomes of the recipient population are replaced by the donor alleles, while mitochondrial genomes retain the original ( gray) genetic components. However, if the replacement is not complete, we would expect that the X chromosome would retain more original genetic components than the autosomes.

Figure 2
Mitochondrial genealogy, including publicly available mitochondrial genome sequences.
Bootstrap percentile values are shown near the nodes.

Figure 3
Neighbor-joining tree constructed using the distances between nuclear genomes. All

Figure 5
The values of f4 statistics on autosomes ( red) and the X chromosome . Negative f4 statistics indicate that B is more closely related to STM than to ASM. B) f4(OLB, ASM; CMA1, D). Negative f4 indicates that ASM is more closely related to CMA1 than to D. C) f4(OLB, B; RMC, RMI). Negative f4 statistics indicate that B is more closely related to RMC than to RMI.

Figure 6
Admixture graph of the mulatta-group species and CMM. OLB was used as the outgroup.
The numbers along the branches The admixture of the hybrid lineage ( node L) and a population diverged from RMI ( node N) generated the ancestral population of RMC. Note that the admixture fraction of node N is much larger on autosomes than on X chromosomes