The complete sequence and comparative analysis of ape sex chromosomes.

Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility1. The X chromosome is vital for reproduction and cognition2. Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species.

Partitioning variance in a signaling trade-off under sexual selection reveals among-individual covariance in trait allocation.

Understanding the evolution of traits subject to trade-offs is challenging because phenotypes can (co)vary at both the among- and within-individual levels. Among-individual covariation indicates consistent, possibly genetic, differences in how individuals resolve the trade-off, while within-individual covariation indicates trait plasticity. There is also the potential for consistent among-individual differences in behavioral plasticity, although this has rarely been investigated. We studied the sources of (co)variance in two characteristics of an acoustic advertisement signal that trade-off with one another and are under sexual selection in the gray treefrog, Hyla chrysoscelis: call duration and call rate. We recorded males on multiple nights calling spontaneously and in response to playbacks simulating different competition levels. Call duration, call rate, and their product, call effort, were all repeatable both within and across social contexts. Call duration and call rate covaried negatively, and the largest covariance was at the among-individual level. There was extensive plasticity in calling with changes in social competition, and we found some evidence for among-individual variance in call rate plasticity. The significant negative among-individual covariance in trait values is perpendicular to the primary direction of sexual selection in this species, indicating potential limits on the response to selection.

Gut microbiota of skywalker hoolock gibbons (Hoolock tianxing) from different habitats and in captivity: Implications for gibbon health.

The gut microbiota plays an integral role in the metabolism and immunity of animal hosts, and provides insights into the health and habitat assessment of threatened animals. The skywalker hoolock gibbon (Hoolock tianxing) is a newly described gibbon species, and is considered an endangered species. Here, we used 16S rRNA amplicon sequencing to describe the fecal bacterial community of skywalker hoolock gibbons from different habitats and in captivity. Fecal samples (n = 5) from two captive gibbons were compared with wild populations (N = 6 gibbons, n = 33 samples). At the phylum level, Spirochetes, Proteobacteria, Firmicutes, Bacteroidetes dominated in captive gibbons, while Firmicutes, Bacteroidetes, and Tenericutes dominated in wild gibbons. At the genus level, captive gibbons were dominated by Treponema-2, followed by Succinivibrio and Cerasicoccus, while wild gibbons were dominated by Anaeroplasma, Prevotellaceae UCG-001, and Erysipelotrichaceae UCG-004. Captive rearing was significantly associated with lower taxonomic alpha-diversity, and different relative abundance of some dominant bacteria compared to wild gibbons. Predicted Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that captive gibbons have significantly lower total pathway diversity and higher relative abundance of bacterial functions involved in "drug resistance: antimicrobial" and "carbohydrate metabolism" than wild gibbons. This study reveals the potential influence of captivity and habitat on the gut bacterial community of gibbons and provides a basis for guiding the conservation management of captive populations.

Dispersal and female philopatry in a long-term, stable, polygynous gibbon population: Evidence from 16 years field observation and genetics.

Gibbons are generally reported to live in small socially monogamous family groups in which both sexes disperse when they reach maturity. For the first time, we documented the dispersal pattern in a population of gibbons living in stable polygynous groups (Nomascus concolor) integrating 16 years' field observation and genetic information from fecal DNA. All subadult males except for one dispersed at 9.8 ± 1.4 years of age (range: 8-12, N = 10). The last male remained in his natal group and obtained the breeding position at age 11 by evicting the original dominant male. Females reached sexual maturity (as evidenced by the change in body color from black to yellow) at 8 years (N = 4). Three of them dispersed and one obtained a position as a breeding female and bred in her natal group. We also observed one female returning to her natal group with her infant after her presumed father was taken over by a neighboring male. We identified only three mtDNA haplotypes from 22 individuals at Dazhaizi. Individuals in one group shared the same haplotype, with only one exception. Genetic results showed that the two breeding females were mother-daughter pairs in all three study groups at the end of this study, implying some degree of female philopatry. We argue that in the case of black crested gibbons, dispersal decisions appear to represent highly opportunistic events in response to reproductive opportunities in their natal and neighboring groups.

Functional characterization of enhancer evolution in the primate lineage.

BACKGROUND: Enhancers play an important role in morphological evolution and speciation by controlling the spatiotemporal expression of genes. Previous efforts to understand the evolution of enhancers in primates have typically studied many enhancers at low resolution, or single enhancers at high resolution. Although comparative genomic studies reveal large-scale turnover of enhancers, a specific understanding of the molecular steps by which mammalian or primate enhancers evolve remains elusive. RESULTS: We identified candidate hominoid-specific liver enhancers from H3K27ac ChIP-seq data. After locating orthologs in 11 primates spanning around 40 million years, we synthesized all orthologs as well as computational reconstructions of 9 ancestral sequences for 348 active tiles of 233 putative enhancers. We concurrently tested all sequences for regulatory activity with STARR-seq in HepG2 cells. We observe groups of enhancer tiles with coherent trajectories, most of which can be potentially explained by a single gain or loss-of-activity event per tile. We quantify the correlation between the number of mutations along a branch and the magnitude of change in functional activity. Finally, we identify 84 mutations that correlate with functional changes; these are enriched for cytosine deamination events within CpGs. CONCLUSIONS: We characterized the evolutionary-functional trajectories of hundreds of liver enhancers throughout the primate phylogeny. We observe subsets of regulatory sequences that appear to have gained or lost activity. We use these data to quantify the relationship between sequence and functional divergence, and to identify CpG deamination as a potentially important force in driving changes in enhancer activity during primate evolution.

Hepatic expression of cytochrome P450 enzymes in non-human primate species.

Cytochromes P450 (P450) largely remain to be characterized in great apes. Comparative immunochemical detection of drug metabolizing forms of P450s 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4A, and 4F in liver microsomes from chimpanzees, gorillas, orangutans, gibbons, cynomolgus and rhesus macaques, and common marmosets were carried out.

Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes.

Positive (adaptive) selection has recently been implied in human superoxide dismutase 1 (SOD1), a highly abundant antioxidant protein with energy signaling and antiaging functions, one of very few examples of direct selection on a human protein product (exon); the molecular drivers of this selection are unknown. We mapped 30 extant SOD1 sequences to the recently established mammalian species tree and inferred ancestors, key substitutions, and signatures of selection during the protein's evolution. We detected elevated substitution rates leading to great apes (Hominidae) at ~1 per 2 million years, significantly higher than in other primates and rodents, although these paradoxically generally evolve much faster. The high evolutionary rate was partly due to relaxation of some selection pressures and partly to distinct positive selection of SOD1 in great apes. We then show that higher stability and net charge and changes at the dimer interface were selectively introduced upon separation from old world monkeys and lesser apes (gibbons). Consequently, human, chimpanzee and gorilla SOD1s have a net charge of -6 at physiological pH, whereas the closely related gibbons and macaques have -3. These features consistently point towards selection against the malicious aggregation effects of elevated SOD1 levels in long-living great apes. The findings mirror the impact of human SOD1 mutations that reduce net charge and/or stability and cause ALS, a motor neuron disease characterized by oxidative stress and SOD1 aggregates and triggered by aging. Our study thus marks an example of direct selection for a particular chemical phenotype (high net charge and stability) in a single human protein with possible implications for the evolution of aging.

Description of a new species of Hoolock gibbon (Primates: Hylobatidae) based on integrative taxonomy.

We describe a species of Hoolock gibbon (Primates: Hylobatidae) that is new to science from eastern Myanmar and southwestern China. The genus of hoolock gibbons comprises two previously described living species, the western (Hoolock hoolock) and eastern hoolock (H. leuconedys) gibbons, geographically isolated by the Chindwin River. We assessed the morphological and genetic characteristics of wild animals and museum specimens, and conducted multi-disciplinary analyses using mitochondrial genomic sequences, external morphology, and craniodental characters to evaluate the taxonomic status of the hoolock population in China. The results suggest that hoolocks distributed to the east of the Irrawaddy-Nmai Hka Rivers, which were previously assigned to H. leuconedys, are morphologically and genetically distinct from those to the west of the river, and should be recognized as a new species, the Gaoligong hoolock gibbon or skywalker hoolock gibbon (H. tianxing sp. nov.). We consider that the new species should be categorized as Endangered under IUCN criteria. The discovery of the new species focuses attention on the need for improved conservation of small apes, many of which are in danger of extinction in southern China and Southeast Asia.

Genetic comparisons yield insight into the evolution of enamel thickness during human evolution.

Enamel thickness varies substantially among extant hominoids and is a key trait with significance for interpreting dietary adaptation, life history trajectory, and phylogenetic relationships. There is a strong link in humans between enamel formation and mutations in the exons of the four genes that code for the enamel matrix proteins and the associated protease. The evolution of thick enamel in humans may have included changes in the regulation of these genes during tooth development. The cis-regulatory region in the 5' flank (upstream non-coding region) of MMP20, which codes for enamelysin, the predominant protease active during enamel secretion, has previously been shown to be under strong positive selection in the lineages leading to both humans and chimpanzees. Here we examine evidence for positive selection in the 5' flank and 3' flank of AMELX, AMBN, ENAM, and MMP20. We contrast the human sequence changes with other hominoids (chimpanzees, gorillas, orangutans, gibbons) and rhesus macaques (outgroup), a sample comprising a range of enamel thickness. We find no evidence for positive selection in the protein-coding regions of any of these genes. In contrast, we find strong evidence for positive selection in the 5' flank region of MMP20 and ENAM along the lineage leading to humans, and in both the 5' flank and 3' flank regions of MMP20 along the lineage leading to chimpanzees. We also identify putative transcription factor binding sites overlapping some of the species-specific nucleotide sites and we refine which sections of the up- and downstream putative regulatory regions are most likely to harbor important changes. These non-coding changes and their potential for differential regulation by transcription factors known to regulate tooth development may offer insight into the mechanisms that allow for rapid evolutionary changes in enamel thickness across closely-related species, and contribute to our understanding of the enamel phenotype in hominoids.

Evolutionary origin of higher-order repeat structure in alpha-satellite DNA of primate centromeres.

Alpha-satellite DNA (AS) is a main DNA component of primate centromeres, consisting of tandemly repeated units of ~170 bp. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, in which a block of multiple repeat units forms a larger repeat unit and the larger units are repeated tandemly. The presence of HOR in AS is widely thought to be unique to hominids (family Hominidae; humans and great apes). Recently, we have identified an HOR-containing AS in the siamang, which is a small ape species belonging to the genus Symphalangus in the family Hylobatidae. This result supports the view that HOR in AS is an attribute of hominoids (superfamily Hominoidea) rather than hominids. A single example is, however, not sufficient for discussion of the evolutionary origin of HOR-containing AS. In the present study, we developed an efficient method for detecting signs of large-scale HOR and demonstrated HOR of AS in all the three other genera. Thus, AS organized into HOR occurs widely in hominoids. Our results indicate that (i) HOR-containing AS was present in the last common ancestor of hominoids or (ii) HOR-containing AS emerged independently in most or all basal branches of hominoids. We have also confirmed HOR occurrence in centromeric AS in the Hylobatidae family, which remained unclear in our previous study because of the existence of AS in subtelomeric regions, in addition to centromeres, of siamang chromosomes.

Cytogenetic studies of small ape (Hylobatidae) chromosomes.

Each genus of small apes has a highly distinctive karyotype (karyomorph) at every level of cytogenetic analysis. Early workers using classical staining and banding had problems integrating the karyolocial data with that of other primates. Chromosome painting allowed syntenic homology maps to be constructed for each of the four karyomorphs (2n = 38, 44, 50 and 52). They revealed that the great apes and Old World monkeys had strongly conserved karyotypes while those of small apes were highly rearranged. However, they provided contradictory phylogenetic results to other bio-molecular tree of small ape evolution. More recently BAC-FISH investigations using a panel of about 900 BACs defined each breakpoint by spanning or flanking BAC clones The syntenic map was refined and now includes small segments of homology which had previously gone undected, marker order (synteny block orientation) and the location of ancestral and Evolutionarily New Centromeres. However, the BAC-FISH data similar to other biomolecular methods used up to now could not resolve the phylogenetic tree of hylobatids. These difficulties may be explained by the rapid divergence of crown hylobatids, reticulate evolution and incomplete lineage sorting. The lack of significant cytogenetic landmarks at the nodes of the gibbon tree could indicate that chromosomal rearrangements did not play a primary role in hylobatid speciation.

Inferring the evolutionary histories of divergences in Hylobates and Nomascus gibbons through multilocus sequence data.

BACKGROUND: Gibbons (Hylobatidae) are the most diverse group of living apes. They exist as geographically-contiguous species which diverged more rapidly than did their close relatives, the great apes (Hominidae). Of the four extant gibbon genera, the evolutionary histories of two polyspecific genera, Hylobates and Nomascus, have been the particular focus of research but the DNA sequence data used was largely derived from the maternally inherited mitochondrial DNA (mtDNA) locus. RESULTS: To investigate the evolutionary relationships and divergence processes of gibbon species, particularly those of the Hylobates genus, we produced and analyzed a total of 11.5 kb DNA of sequence at 14 biparentally inherited autosomal loci. We find that on average gibbon genera have a high average sequence diversity but a lower degree of genetic differentiation as compared to great ape genera. Our multilocus species tree features H. pileatus in a basal position and a grouping of the four Sundaic island species (H. agilis, H. klossii, H. moloch and H. muelleri). We conducted pairwise comparisons based on an isolation-with-migration (IM) model and detect signals of asymmetric gene flow between H. lar and H. moloch, between H. agilis and H. muelleri, and between N. leucogenys and N. siki. CONCLUSIONS: Our multilocus analyses provide inferences of gibbon evolutionary histories complementary to those based on single gene data. The results of IM analyses suggest that the divergence processes of gibbons may be accompanied by gene flow. Future studies using analyses of multi-population model with samples of known provenance for Hylobates and Nomascus species would expand the understanding of histories of gene flow during divergences for these two gibbon genera.

Phylogenetic separation in limb use in captive gibbons (Hylobatidae): a comparison across the primate order.

Although there have been few studies of self-scratching in primates, some have reported distinct differences in whether hands or feet are used, and these variations seem to reflect the evolutionary history of the Order. Monkeys and prosimians use both hands and feet to self-scratch while African great apes use hands almost exclusively. Gibbons represent an evolutionary divergence between monkeys and great apes and incidental observations at the Gibbon Conservation Center pointed to a difference in self-scratching among the four extant gibbon genera (Hoolock, Nomascus, Symphalangus, and Hylobates). To validate and further explore these preliminary observations, we collected systematic data on self-scratching from 32 gibbons, including nine species and all four genera. To supplement gibbon data, we also collected self-scratching information from 18 great apes (four species), five prosimians (two species), 26 New World Monkeys (nine species) and 20 Old World Monkeys (seven species). All monkeys and some prosimians used both hands and feet to self-scratch, whereas one prosimian species used only feet. All African great apes used hands exclusively (orangutans were an exception displaying occasional foot-use). This appears to represent a fundamental difference between monkeys and great apes in limb use. Interestingly, there was a clear difference in self-scratching between the four gibbon genera. Hylobates and Symphalangus self-scratched only with hands (like all African great apes), while Hoolock and Nomascus self-scratched with both hands and feet (like monkeys and prosimians). This difference in gibbon behavior may reflect the evolutionary history of gibbons as Hoolock and Nomascus are thought to have evolved before both Hylobates and Symphalangus. What evolutionary pressures led to this divergent pattern is currently opaque; however, this shift in limb preference may result from niche separation across the order facilitating differences in the behavioral repertoire associated with hind and forelimbs.

Evolutionary molecular cytogenetics of catarrhine primates: past, present and future.

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.

Extrapair paternity in golden-cheeked gibbons (Nomascus gabriellae) in the secondary lowland forest of Cat Tien National Park, Vietnam.

We observed 18 groups of golden-cheeked gibbons (Nomascus gabriellae) in the secondary lowland forest in Cat Tien National Park from January 2004 to December 2005 to obtain information about the social organization of this little-studied species, in an area where the population is recovering through increased protection and forest regeneration. DNA from faecal samples of 10 infants and juveniles identified 1 case of extrapair paternity. DNA from faecal samples of 18 adults from three communities revealed the majority of adults sampled of both sexes to be related to adults in neighbouring territories. Overall, the indications are that in this empty habitat, gibbons appear to be able to establish territories adjacent to those of their kin.

A comparative study of crested gibbons (Nomascus).

Crested gibbons (Nomascus) are in the rarest genus of the family Hylobatidae, with the Hainan gibbon (Nomascus hainanus) being the rarest primate in the world. In the past, the number of species in this genus has been at the center of much controversy, in part, because their color changes during immaturity as well as other factors, such as physical similarities in genitalia, creating difficulties in accurately determining the sex of individuals. Furthermore, owing to their rarity, illusiveness, and the rough terrain that comprises their native habitat, Nomascus is one of the least studied Hylobatidae. This article represents the most comprehensive dissemination of visual characteristics of the genus Nomascus to assist in the accurate identification of captive and wild crested gibbons. Through differences in pelage color, skeletal anatomy, dentition, vocalizations, behavior, distribution, and genetic studies, we are able to determine more accurately whether or not a subspecies should be elevated to species level. From the current data, there are six species and one subspecies in the genus Nomascus. However, reports of a recently identified light-cheeked gibbon (Nomascus sp.) in northeast Cambodia, Central Vietnam, and South Lao PDR, will add additional taxa to this genus.

Evolution of the hepcidin gene in primates.

BACKGROUND: Hepcidin/LEAP-1 is an iron regulatory hormone originally identified as an antimicrobial peptide. As part of a systematic analysis of the evolution of host defense peptides in primates, we have sequenced the orthologous gene from 14 species of non-human primates. RESULTS: The sequence of the mature peptide is highly conserved amongst all the analyzed species, being identical to the human one in great apes and gibbons, with a single residue conservative variation in Old-World monkeys and with few substitutions in New-World monkeys. CONCLUSION: Our analysis indicates that hepcidin's role as a regulatory hormone, which involves interaction with a conserved receptor (ferroportin), may result in conservation over most of its sequence, with the exception of the stretch between residues 15 and 18, which in New-World monkeys (as well as in other mammals) shows a significant variation, possibly indicating that this structural region is involved in other functions.

Evolutionary history of chromosome 11 featuring four distinct centromere repositioning events in Catarrhini.

Panels of BAC clones used in FISH experiments allow a detailed definition of chromosomal marker arrangement and orientation during evolution. This approach has disclosed the centromere repositioning phenomenon, consisting in the activation of a novel, fully functional centromere in an ectopic location, concomitant with the inactivation of the old centromere. In this study, appropriate panels of BAC clones were used to track the chromosome 11 evolutionary history in primates and nonprimate boreoeutherian mammals. Chromosome 11 synteny was found to be highly conserved in both primate and boreoeutherian mammalian ancestors. Amazingly, we detected four centromere repositioning events in primates (in Old World monkeys, in gibbons, in orangutans, and in the Homo-Pan-Gorilla (H-P-G) clade ancestor), and one in Equidae. Both H-P-G and Lar gibbon novel centromeres were flanked by large duplicons with high sequence similarity. Outgroup species analysis revealed that this duplicon was absent in phylogenetically more distant primates. The chromosome 11 ancestral centromere was probably located near the HSA11q telomere. The domain of this inactivated centromere, in humans, is almost devoid of segmental duplications. An inversion occurred in chromosome 11 in the common ancestor of H-P-G. A large duplicon, again absent in outgroup species, was found located adjacent to the inversion breakpoints. In Hominoidea, almost all the five largest duplicons of this chromosome appeared involved in significant evolutionary architectural changes.

A complete species-level phylogeny of the Hylobatidae based on mitochondrial ND3-ND4 gene sequences.

The Hylobatidae (gibbons) are among the most endangered primates and their evolutionary history and systematics remain largely unresolved. We have investigated the species-level phylogenetic relationships among hylobatids using 1257 bases representing all species and an expanded data set of up to 2243 bases for select species from the mitochondrial ND3-ND4 region. Sequences were obtained from 34 individuals originating from all 12 recognized extant gibbon species. These data strongly support each of the four previously recognized clades or genera of gibbons, Nomascus, Bunopithecus, Symphalangus, and Hylobates, as monophyletic groups. Among these clades, there is some support for either Bunopithecus or Nomascus as the most basal, while in all analyses Hylobates appears to be the most recently derived. Within Nomascus, Nomascus sp. cf. nasutus is the most basal, followed by N. concolor, and then a clade of N. leucogenys and N. gabriellae. Within Hylobates, H. pileatus is the most basal, while H. moloch and H. klossii clearly, and H. agilis and H. muelleri likely form two more derived monophyletic clades. The segregation of H. klossii from other Hylobates species is not supported by this study. The present data are (1) consistent with the division of Hylobatidae into four distinct clades, (2) provide the first genetic evidence for all the species relationships within Nomascus, and (3) call for a revision of the current relationships among the species within Hylobates. We propose a phylogenetic tree as a working hypothesis against which intergeneric and interspecific relationships can be tested with additional genetic, morphological, and behavioral data.

Evolution of the beta defensin 2 gene in primates.

With the aim of further investigating the molecular evolution of beta defensin genes, after having analysed beta defensin 1 (DEFB1) in humans and several nonhuman primate species, we have studied the evolution of the beta defensin 2 gene (DEFB2), which codifies for a peptide with antimicrobial and chemoattractant activity, in humans and 16 primate species. We have found evidence of positive selection during the evolution of orthologous DEFB2 genes at two points on a phylogenetic tree relating these primates: during the divergence of the platyrrhines from the catarrhines and during the divergence of the Cercopithecidae from the Hylobatidae, Great Apes and humans. Furthermore, amino acid variations in Old World Monkeys seem to centre either on residues that are involved in oligomerisation in the human molecule, or that are conserved (40-80%) in beta-defensins in general. It is thus likely that these variations affect the biological function of the molecules and suggest that their synthesis and functional analysis might reveal interesting new information as to their role in innate immunity.