Abstract Diet is a powerful evolutionary force for species adaptation and diversification. Acari is one of the most abundant clades of Arachnida, exhibiting diverse dietary types, while the underlying genetic adaptive mechanisms are not fully understood. Based on comparative analyses of 15 Acari genomes, we found genetic bases for three specialized diets. Herbivores experienced stronger selection pressure than other groups; the olfactory genes and gene families involving metabolizing toxins showed strong adaptive signals. Genes and gene families related to anticoagulation, detoxification, and haemoglobin digestion were found to be under strong selection pressure or significantly expanded in the blood-feeding species. Lipid metabolism genes have a faster evolutionary rate and been subjected to greater selection pressures in fat-feeding species; one positively selected site in the fatty-acid amide hydrolases 2 gene was identified. Our research provides a new perspective for the evolution of Acari and offers potential target loci for novel pesticide development. Subject terms: Genomics, Evolutionary genetics __________________________________________________________________ Liu et al. present a comparative analysis of 15 genomes from mites and identify genetic signatures for diet specialisation. Different gene families and selective pressures were revealed for herbivorous, haematophagous and fat-feeding species. Introduction Diet is one of the most fundamental aspects of an animal’s biology and is a powerful evolutionary force for species adaptation and diversification^[34]1–[35]3. Different diets have been acknowledged to generate various physiological, biochemical, and morphological adaptations^[36]4. With the development of genome sequencing, the genetic basis of dietary adaptation has been revealed gradually, providing new and valuable insight into evolutionary biology^[37]5–[38]8. The abundant species diversity of mites and ticks (Arachnida: Acari) has inspired acarologists and ecologists to understand their evolutionary processes and ecological characteristics for decades^[39]9. Approximately 55,000 species have been described^[40]10, and the total species number is estimated to be much larger^[41]9,[42]10. It was reported that the diverse lifestyles in Acari (e.g., free-living, symbiotic, and parasitic) serve as an important driver of its species diversity^[43]11. The dietary lifestyles of Acari range from predatory to parasitic^[44]9. Compared with spiders with a predatory diet, Acari exhibit several new diets, such as herbivory and parasitism (e.g., blood and fat bodies), as well as some transitional states such as the consumption of skin exudates, decomposing biomass, and fungi^[45]9,[46]12. These different specialized diets have generated a number of physiological, biochemical, and morphological adaptations^[47]4. For example, herbivorous mites have developed a long and stout stylet-like structure for feeding on plant juice^[48]13,[49]14. A key mechanism of phenotypic adaptation is genetic adaptation, including changes in functional genes, metabolic and regulatory pathways, and even amino acids^[50]5,[51]6,[52]15. However, the genetic mechanisms underlying the dietary adaptation of Acari have not yet been systematically discussed in a comparative manner. According to the phylogeny of Arachnida^[53]16,[54]17, Acari consists of two major clades: Acariformes and Parasitiformes^[55]18. In Acariformes, some mites maintained predatory diets, while some obtained transitional, herbivorous (e.g., Tetranychus urticae) or blood sucking (e.g., Leptotrombidium deliense) diets^[56]19,[57]20. In Parasitiformes, ticks (Ixodida) became obligate hematophages and some mites also developed parasitic diets, feeding on blood and fat bodies, respectively^[58]12,[59]21. The distributions of different diets across the phylogeny of Acari provide an opportunity to understand the genetic mechanisms of dietary adaptation and evolution. In the current study, we performed comparative analyses of genomes from fifteen Acari species in four major orders and with five kinds of diets. We constructed a well-resolved fossil-calibrated phylogenetic tree and revealed the genomic evolution of Acari. Furthermore, we investigated the genetic adaptation associated with three specialized diets, blood sucking, herbivory, and fat feeding, and found important sites and pathways that underwent adaptive convergent evolution. Our findings, with further experimental validation, can be used as potential targets for drug and pesticide research to control herbivorous and parasitic mites. Results and discussion A total of sixteen Arachnida genomes, including those of one tick, fourteen mites, and the velvet spider as an outgroup, were obtained from GenBank (details in “Methods”). The sixteen species displayed all five kinds of diets, including three predacious species, two herbivorous species, three fat-feeding species, three blood-feeding species, and five transitional species (Table [60]1). Six genomes were reannotated (marked in Table [61]1) using ab initio prediction and homology prediction methods together (details in “Methods”). To evaluate genome completeness, we examined the genome and genes by Benchmarking Universal Single-Copy Orthologs (BUSCO)^[62]22. We observed high BUSCO scores of genome completeness (average 90.9%) and gene completeness (average 79.9%) (Supplementary Table [63]1). This result suggested that the assemblies of the sixteen genomes were of high quality for downstream comparative analyses. Table 1. Genomes information used in this study. Latin name Common name GenBank AssemblyAccession Total size of contigs Contig N50 Gene number Diet Taxonomy Ixodes scapularis^[64]74 Black-legged tick GCF_002892825.2 2,081,329,876 835,681 24,489 Blood sucking Parasitiformes Metaseiulus occidentalis^[65]75 Predatory mite GCF_000255335.1 151,323,873 200,706 11,603 Predation Parasitiformes Dermanyssus gallinae^[66]76^* Roost mite GCA_003439945.1 959,010,206 278,630 42,159 Blood sucking Parasitiformes Tropilaelaps mercedesae^[67]77 Honeybee mite GCA_002081605.1 326,213,305 13,741 15,190 Fat feeding Parasitiformes Varroa destructor^[68]78 Honeybee mite GCF_002443255.1 368,670,960 201,886 10,241 Fat feeding Parasitiformes Varroa jacobsoni^[69]78 Honeybee mite GCF_002532875.1 365,177,116 96,030 10,724 Fat feeding Parasitiformes Steganacarus magnus^[70]79^* Oribatid mite GCA_000988885.1 112,750,608 1727 9990 Transitional Acariformes Hypochthonius rufulus^[71]79^* Soil mite GCA_000988845.1 171,814,378 3254 6285 Transitional Acariformes Sarcoptes scabiei^[72]80 Scabies mite GCA_000828355.1 56,251,741 11,383 10,644 Transitional Acariformes Dermatophagoides farinae^[73]81^* House dust mite GCA_002085665.1 91,934,661 188,869 15,394 Transitional Acariformes Psoroptes ovis^[74]82^* sheep scab mite GCA_002943765.1 63,214,126 2,279,290 12,041 Transitional Acariformes Leptotrombidium deliense^[75]83 Chigger GCA_003675905.2 117,276,895 1422 15,096 Blood sucking Acariformes Dinothrombium tinctorium^[76]83 Velvet mites GCA_003675995.1 180,156,552 16,116 19,258 Predation Acariformes Brevipalpus yothersi^[77]84^* Flat mite GCA_003956705.1 70,567,388 56,520 8,245 Herbivory Acariformes Tetranychus urticae^[78]33 Spider mite GCF_000239435.1 89,613,205 212,780 18,414 Herbivory Acariformes Stegodyphus mimosarum^[79]85 Velvet spider GCA_000611955.2 2,694,371,924 46,340 27,235 Predation Araneae [80]Open in a new tab Star-tagged species indicate that their genomes have been reannotated To reveal the genomic signatures of dietary adaptations in Acari, we constructed a genome-wide phylogenetic tree based on 48,831 nucleotides. Based on three fossil calibration points and a relaxed molecular clock, the divergence time between Acari and spiders was estimated to be ~477.6 million years ago (Mya) (Fig. [81]1). Moreover, the divergence between Sarcoptiformes and Trombidiformes was estimated to be ~321.8 Mya, and the divergence between Ixodida and Mesostigmata occurred ~336 Mya (Fig. [82]1). The phylogenetic tree and time estimates of key nodes were consistent with those in recent studies^[83]16,[84]23–[85]25. Fig. 1. The genome-wide phylogenetic tree of Arachnida. [86]Fig. 1 [87]Open in a new tab The divergence time was estimated based on a maximum likelihood phylogenomic tree of 16 Arachnida species and three fossil calibrations. All nodes have 100% support according to 1,000 bootstraps. The estimated divergence times are displayed with 95% confidence intervals (in square brackets). Blood-sucking species in red; fat-feeding species in olive; herbivorous species in green; predatory species in blue; and others/transitional in orange. The same colour theme is used in the other figures. The numbers to the right of the species indicate the records of gene family expansions and contractions, with red for expansions and blue for contractions. Relaxation of selective constraints on and loss of function of protein-coding genes may occur during dietary shifts^[88]6,[89]26. A total of 1,210 pseudogenes were identified across all species (Fig. [90]2a, Supplementary Data [91]1). Blood-feeding species with larger genomes had a higher ratio of pseudogenes (Fig. [92]2a), supporting the idea that pseudogenes act as a determinant of genome size evolution^[93]27. The functions of these pseudogenes, most of which were involved in signal transduction, transport, catabolism and so on, were similar across species (Fig. [94]2b). A total of 65, 23, 54 and 27, and 29 pseudogenes were detected to be common in the groups fed the five diets, predation, blood, plant, fat, and others (transitional type), respectively (Fig. [95]2a, Supplementary Data [96]2). Among these pseudogenes, the genes for ribonuclease H were pseudogenized in both the blood-sucking group and fat-feeding group (Supplementary Data [97]2), which may be related to their parasitic lifestyles. Interestingly, no pseudogenes were shared across all dietary groups, implying that different patterns occurred during the dietary shift (Supplementary Data [98]2). Since the pseudogenes are nonfunctional, subsequent analyses based on functional genes were carried out after removing the pseudogenes. Fig. 2. Pseudogene and PSG profiles of different dietary groups. [99]Fig. 2 [100]Open in a new tab a The overall numbers of pseudogenes detected in each group are shown in different colours reference to left y-axis, and the numbers of shared pseudogenes in each group are shown in black. The pseudogenes in each group are divided into three types, namely, frame-shift mutation, premature-stop codon, and both, and the ratios of types are displayed by lines reference to right y-axis. b The functional classification of the pseudogenes in each group. c KEGG enrichment analysis of the PSGs. d PSGs generated by the orthologous genes annotated in at least two species in each dietary group. The number in parentheses represents the number of PSGs in the group. The dot plot shows the numbers of positively selected sites across four different dietary groups. The x-axis indicates the number of loci detected as being under selection pressure, and the y-axis indicates the number of positively selected loci with a BEB posterior probability greater than 95% in PAML. PSG positively selected gene, KEGG Kyoto Encyclopedia of Genes and Genomes, BEB Bayesian empirical Bayes. To detect signals of positive selection in Acari with specialized diets, we performed phylogenetic analysis by maximum likelihood (PAML) branch-site model (model = 2, and NSsites = 2) tests for the single-copy orthologous genes. As a result, 530 positively selected genes (PSGs) were identified, and 291, 56, 73, and 110 PSGs were detected for the plant, predation, blood, and fat diets, respectively (Supplementary Data [101]2). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the PSGs were partly correlated with the ability to digest different food types, such as Porphyrin and chlorophyll metabolism in the plant dietary group and Arachidonic acid metabolism in the fat dietary group (Fig. [102]2c, Supplementary Data [103]2). Gene ontology (GO) enrichment analysis also revealed biological processes correlated with different diets, such as organonitrogen metabolism in the herbivorous group (Supplementary Fig. [104]1 and Supplementary Data [105]2). To compare the overall pressure of natural selection on different food preferences, a total of