Comparative genomics reveal signatures of ecological specialization in the striped ambrosia beetle Trypodendron lineatum

Comparative genomics reveal signatures of ecological specialization in the striped ambrosia beetle Trypodendron lineatum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative genomics reveal signatures of ecological specialization in the striped ambrosia beetle Trypodendron lineatum Zaide Montes-Ortiz, Daniel Powell, Heiko Vogel, Christer Löfstedt, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8230855/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Beetles (Coleoptera) exhibit remarkable dietary versatility, which may drive genomic innovations. Ambrosia beetles (Curculionidae: Scolytinae) have evolved specific feeding habits and intricate relationships with symbiotic fungi. The striped ambrosia beetle Trypodendron lineatum is a pest of conifers, relying on its obligate nutritional mutualist Phialophoropsis ferruginea for survival. The beetles cultivate the fungi inside their galleries in the tree’s xylem, with the fungi serving as their sole food source. We hypothesize that this lifestyle is associated with genomic signatures that may reflect important adaptations. Hence, we performed a comparative genomic analysis between T. lineatum and nine other beetle species, including related scolytine bark beetles, to uncover genomic signatures of this specialization, focusing on gene families involved in e.g. digestion, detoxification, and immunity. Results The small genome of T. lineatum (74.4–83.6 Mb) exhibits comparatively low levels of repetitive DNA (19.9%), including a reduced proportion of transposable elements, and unusually short introns. Annotation generated 14,830 high-quality gene predictions, most of which were supported by transcript evidence or functional domains. Comparative orthology analysis identified 13,896 orthogroups, with T. lineatum showing 78 species-specific orthogroups and a set of gene family changes which may reflect its ecological specializations. Thirty-three T. lineatum gene families showed significant size changes, including 16 expansions and 17 contractions. Notably, gene families associated with digestion, detoxification, and immunity were contracted. These included glycoside hydrolase 28, cytochrome P450, serpin, and trypsin families, suggesting reduced reliance on plant-based digestion and broad-spectrum immune defenses. In contrast, expansions in the THAP domain and CD80-like immunoglobulin domain families indicate selective retention and diversification of genes involved in genomic regulation and immune recognition. Conclusions Our results suggests that the genome of T. lineatum is streamlined, characterized by a low repeat content and compact gene architecture. The observed contractions in key gene families involved in plant digestion, detoxification, and immunity likely represent genomic signatures of its obligate mutualistic specialization and narrow ecological niche. Our findings provide the first insights into the genomic adaptations of fungus-farming ambrosia beetles, suggesting that co-evolved insect-microbe mutualisms may lead to reductions in a variety of insect gene families. Coleoptera Curculionidae Scolytinae Symbiosis Genome annotation Gene family evolution Immune gene contraction Detoxification Figures Figure 1 Figure 2 Introduction Beetles (Coleoptera) represent one of the most species-rich and ecologically diverse lineages in the animal kingdom, comprising over 380,000 described species across a wide range of terrestrial and freshwater ecosystems [ 1 , 2 ]. Moreover, their evolutionary success has been attributed in part to their remarkable diversity of feeding strategies, allowing them to exploit a wide variety of ecological niches [ 3 , 4 ]. These include phytophagy (feeding on plants), saproxyly (feeding on decaying wood) [ 5 ], predation [ 6 ], and mycophagy (fungus-feeding) [ 7 , 8 ]. Importantly, several of these feeding strategies are often accompanied by symbiotic relationships with microbes [ 9 ]. This dietary versatility has not only fostered extensive diversification but has also driven functional innovations at morphological (e.g., specialized mouthparts) [ 10 ], physiological (e.g., digestive enzymes) [ 11 , 12 ], and genomic (e.g., detoxification-related and chemosensory genes) levels [ 13 – 16 ]. Bark- and ambrosia beetles (family Curculionidae) are both members of the subfamily Scolytinae. However, ambrosia beetles are a polyphyletic group with members also in Platypodinae, and they differ from bark beetles in terms of feeding behavior, larval development, and associations with symbiotic microbes [ 17 , 18 ]. Bark beetles primarily feed on the host tree phloem inoculated with beetle-associated symbiotic fungi. Inside the phloem, their larvae construct individual tunnels away from the maternal gallery, with limited parent-offspring interaction [ 7 , 17 – 19 ]. In contrast, ambrosia beetles bore into the xylem and cultivate obligate mutualistic fungi within their galleries. Rather than feeding on wood directly, they rely on these fungal gardens as their sole nutritional source [ 20 – 22 ]. This tight interdependence exemplifies a co-evolved obligate nutritional mutualism that has shaped the ecological strategies and life histories of ambrosia beetles in fundamentally different ways from their bark beetle relatives [ 7 ]. This symbiotic relationship is maintained through complex mechanisms, including the ability of ambrosia beetles to store and maintain fungal spores in a dormant state within specialized glandular mycangia. This process has likely had a significant evolutionary impact on their immune system and associated genes [ 23 ]. The striped ambrosia beetle, Trypodendron lineatum Olivier, is a significant pest species of Holarctic conifer forests [ 20 , 24 , 25 ]. It targets stressed or dying conifer trees, boring into the xylem to establish larval galleries, which are inoculated with spores of its obligate fungal mutualist, Phialophoropsis ferruginea (Mathiesen-Käärik) (Ascomycota) [ 26 ]. This fungus-farming lifestyle is a prime example of niche construction, enabling T. lineatum to modify its environment effectively [ 27 ]. To find suitable host trees for colonization, volatile organic compounds from decaying conifers, such as α-pinene and ethanol [ 28 ], attract T. lineatum , whereas volatiles from non-host angiosperm trees are avoided [ 20 , 29 ].The beetle also uses the female-produced aggregation pheromone (+)-lineatin (3,3,7-trimethyl-2,9-dioxatricyclononane) [ 30 ], which attracts both sexes to trees. Moreover, the presence of olfactory sensory neurons that specifically respond to volatiles produced by its fungal mutualist highlights a finely tuned sensory system for locating suitable habitats and maintaining the fungal association [ 13 ]. Hence, T. lineatum could be considered to exhibit a narrow ecological niche and strong dependence on both host trees and fungal partners, which makes it an ideal model for studying the genomic consequences of mutualistic specialization, especially in comparison with closely related bark beetle species with different ecological adaptations [ 31 – 33 ]. Whereas large-scale genomic annotation and analyses of the bark beetles Ips typographus L. (Eurasian spruce bark beetle) [ 31 ], Dendroctonus ponderosae Hopkins (mountain pine beetle) [ 33 , 34 ], and Hypothenemus hampei Ferarri (coffee berry borer) [ 32 ] have yielded insights into these species’ ecological specializations, similar analysis has, to the best of our knowledge, never been performed on an ambrosia beetle. In fact, T. lineatum is one of the few ambrosia beetle species with a sequenced genome [ 35 ], making this species a prime target for such analysis. Ecological specialization, particularly in species that engage in obligate mutualisms, is often accompanied by genomic signatures that reflect adaptation to narrow niches [ 3 , 36 ]. A previous study showed that T. lineatum possesses a reduced repertoire of chemoreceptor-coding genes compared to other scolytine beetles, potentially reflecting a streamlined sensory system tailored to its specific associations with its host trees and fungal mutualist [ 35 ]. However, it remains unclear whether such specialization has had broader impacts on other gene families involved in e.g. digestion, detoxification, immunity, or development. To address this, we performed a comparative genomic and orthology analysis of T. lineatum and nine other beetle species, including related scolytine bark beetles, as well as other less related species. We focused on the expansion and contraction of gene families as a proxy for functional adaptation, with the aim of uncovering how ecological constraints and mutualistic strategies have shaped the genome of T. lineatum . Methods Data sources The genome sequencing (EMBL-EBI accession number PRJEB74033; https://www.ebi.ac.uk/ena/browser/view/PRJEB74033 ) and assembly were performed previously (see [ 35 ] for details), which involved extracting genomic DNA from T. lineatum of Swedish origin and sequencing with Oxford Nanopore (~ 190 x genome coverage) and Illumina platforms [ 29 ]. This comprehensive approach resulted in a high-quality assembly, divided between 833 contigs (N50: 915 kbp; longest contig: 4.16 Mbp) [ 35 ] that formed the basis for the present work. Additionally, three distinct datasets were used in this study: (1) a set of target beetle genomes, (2) annotated proteins retrieved from public databases, and (3) whole-body transcriptomes of T. lineatum generated in the present study as well as an antennal transcriptome from [ 35 ] (NCBI accession number PRJNA1126204; https://www.ncbi.nlm.nih.gov/sra/PRJNA1126204 ). In brief, for (1), we downloaded 13 beetle genomes from different families (January 2024) deposited in the public database GenBank from the National Center for Biotechnology Information (NCBI); accession numbers for each species can be found in Additional file 1, Supplementary Table 1 . For (2), we downloaded a non-redundant, well-annotated set of proteins from the RefSeq by using the query “("beetle species"[Organism] OR beetle specie [All Fields]) AND refseq[filter],” where “beetle species” was replaced by each of the ten species used for the orthology analysis. The data from I. typographus , H. hampei , and Callosobruchus maculatus Fabricius (Chrysomelidae) were obtained from public repositories ( Additional file 1, Supplementary Table 1 ). Genome size estimation and identification of telomeres The genome size of T. lineatum was estimated using a k-mer-based approach. Raw sequencing reads were first processed with Trimmomatic (v0.36) for quality control [ 37 ]. K-mer frequency analysis was then performed using Jellyfish (v2.2.10) with a k-mer length of 21, and the resulting distribution was analyzed by GenomeScope (v2.0) to determine genome size, heterozygosity, and repeat content [ 38 , 39 ]. To contextualize the estimated genome size, we used QUAST (v.5.3.0) to compare it with the genome assemblies of 14 other beetle species [ 40 ]. Furthermore, telomeric regions within the T. lineatum genome were searched for using the Telomere Identification toolKit (tidk) [ 41 ] and the FindTelomer.py script (available at https://github.com/JanaSperschneider/FindTelomeres ). We specifically searched for the telomeric repeat motifs TTAGG, TTAGGG, CCCTAA, and TCAGG commonly found in Coleoptera [ 42 ]. Transcriptome sequencing, read mapping, and assembly Adult T. lineatum males and females for transcriptome sequencing were collected from a conifer dominated forest in Tågaröd, South Sweden, during May 2023 using pheromone-baited (Lineatin Kombi dispensers, Witasek, Austria) traps (WitaTrap, 12 funnel size, Witasek). The whole bodies of six females and six males (in two separate samples) were used to isolate total RNA using the RNeasy Minikit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Then, the RNA was DNAse-treated and subjected to library construction using the Illumina TrueSeq stranded mRNA (polyA) kit (Illumina, San Diego, CA, USA). The samples were sequenced on a NovaSeq6000 (NovaSeq Control Software 1.8.1/RTA v3.4.4) with a 151nt(Read1)-19nt(Index1)-10nt(Index2)-151nt(Read2) setup using 'NovaSeqXp' workflow in 'S4' mode flowcell. The raw reads were initially assessed for quality using FastQC (v2.2.1). Adapter trimming and quality filtering were performed using Trimmomatic [ 37 ], followed by a secondary quality assessment with FastQC (v2.2.1) [ 43 ], with summary reports generated by MultiQC [ 44 ]. High-quality reads were mapped to the T. lineatum reference genome using HISAT2 (v2.2.1) [ 45 ]. De novo transcriptome assembly was conducted with Trinity (v2.9.1) [ 46 ]. Additionally, CD-HIT-EST was performed with a sequence identity threshold of 0.98 to reduce redundancy among the assembled transcripts [ 47 ]. Finally, coding regions within the assembled transcripts were predicted using TransDecoder (v5.7.0) (available at https://github.com/TransDecoder/TransDecoder ). Genome annotation and quality assessment The genome of T. lineatum [ 35 ] was annotated using two complementary pipelines: MAKER (v3.01.2) [ 29 ] and BRAKER (v3) [ 48 ]. Before annotation, repetitive elements were identified by constructing an ad hoc repeat library directly from the T. lineatum genome assembly using RepeatModeler (v4.1.0) [ 49 ]. This custom library was combined with the comprehensive Repbase repeat database and subsequently applied with RepeatMasker (v4.0.8) to soft-mask repetitive regions in the genome [ 50 ]. Following the masking step, the MAKER annotation pipeline was executed in four iterative rounds. In the initial round, the annotation process incorporated various sources of evidence data, including transcriptome assemblies derived from whole-body and antennal tissues of T. lineatum , as well as the predicted protein sets from nine coleopteran species ( D. ponderosae , Dendroctonus valens LeConte, H. hampei , I. typographus (all Curculionidae), Anoplophora glabripennis Motschulsky (Cerambycidae), Diabrotica virgifera LeConte, Leptinotarsa decemlineata Say (both Chrysomelidae), Nicrophorus vespilloides Herbst (Silphidae), Tribolium castaneum Herbst (Tenebrionidae)) and Drosophila melanogaster Meigen (Diptera) ( Additional file 1, Supplementary Table 1 ). Additionally, the Insecta_Odb10 database (available at https://busco.ezlab.org/ ), containing 1,367 core genes representing 75 insect species across 14 orders, was included to enhance the accuracy and completeness of the first gene model prediction. In the second round, the gene models generated in the initial round were used as input to train gene prediction software, specifically AUGUSTUS with BUSCO and SNAP. The outputs from these trained predictors were then integrated into MAKER to generate refined second-round gene models, progressively improving annotation quality. Consequently, for the third round, the gene models from the second round served as input, resulting in a new generation of gene models. Finally, a fourth round of annotation was performed by integrating gene predictions generated by the BRAKER3 (v3). These BRAKER3-generated gene models were merged with the MAKER annotation to produce a comprehensive and robust set of final gene annotations. After each round, the Annotation Edit Distance (AED) distribution was calculated. Finally, the redundancy and duplicated sequences were removed from the genome annotation generated with CD-HIT with an identity threshold of 98% [ 47 ], and the assembly quality and completeness assessment were performed using BUSCO with the arthropoda_Odb10 database [ 51 ]. All scripts, parameter files, and detailed commands used in the genome annotation workflow are publicly available at: https://github.com/lachemontes/comparativeGenomics_Tlin . Functional annotation and orthologs gene detection For functional annotation, InterProScan (v2.1.4-2) was used to assign functional categories to the set of predicted protein-coding genes [ 52 ]. The analysis included the following databases: Gene3D, ProSite, Patterns, PANTHER, CDD, Pfam, Phobius, SUPERFAMILY, and TMHMM. In addition, eggNOG-mapper v1.0.3 was used to assign Gene Ontology (GO) terms based on the eggNOG database [ 53 ]. Then, before ortholog detection, a data pre-processing step was performed to improve sequence quality and consistency. Redundant protein sequences were clustered with CD-HIT at a 98% identity threshold, and sequences shorter than 100 amino acids were removed. Subsequently, orthology inference was performed using OrthoFinder (v2.5.2) [ 54 ]. Protein sequences from ten beetle species ( T. lineatum , I. typographus , D. ponderosae , H. hampei , T. castaneum , A. glabripennis , C. maculatus , L. decemlineata , D. virgifera , and Aethina tumida Murray (Nitidulidae)) were included. OrthoFinder was run using gene tree inference (-M msa), multiple sequence alignment (-A mafft), and the default tree inference program. Finally, to assign putative functions to orthogroups, InterProScan (v2.1.4-2) and BLAST searches (e-value < 1e − 5) against the NCBI non-redundant insect database (accessed April 2025) were performed, retaining only non-redundant and significant hits. This functional annotation of orthogroups was critical for subsequent gene family evolution analyses with CAFE, allowing biological interpretation of significantly expanded and contracted gene families [ 55 ]. Gene family evolution: expansions and contractions We used CAFE (v5) with the 13,896 orthogroups identified during the OrthoFinder analysis to investigate the evolutionary dynamics of gene families. Gene family count data from these orthogroups were then reformatted to be compatible with CAFE requirements. In this analysis, we were able to estimate gene turnover rates (λ) for the 10 above-mentioned beetle species from five taxonomic families, to infer ancestral gene counts for each species, and to estimate gene gain/loss rates for each lineage of the phylogeny. To improve phylogenetic inference beyond the default tree construction method (FastTree) used by OrthoFinder, we used the “SpeciesTreeAlignment” output to reconstruct a species tree with IQ-TREE [ 56 ]. The best-fit evolutionary model was selected automatically using ModelFinder [ 57 ], and was evaluated through 1,000 bootstrap iterations. The resulting phylogenetic tree was converted into an ultrametric tree using the ‘ape’ package (v3.0) in R. Divergence time estimates for T. castaneum and I. typographus were obtained from TimeTree5 to calibrate the ultrametric tree (available at https://timetree.org ). Reconstruction of phylogenetic trees For each gene family identified as significantly contracted or expanded, we reconstructed unrooted phylogenetic trees using the approximate maximum-likelihood method implemented in IQ-TREE (v2.4.0) [ 56 ]. The resulting phylogenetic trees ( Additional File 2, Supplementary Figures S2 A-E ) were then visualized and annotated using iTOL (v.7) [ 58 ]. Results and discussion Genome size and telomeric regions The k-mer analysis of raw sequencing reads produced an estimated genome size for T. lineatum ranging from 74.44 Mb to 83.62 Mb, which is smaller than typically observed in other beetle species ( Table 1 ). Heterozygosity was estimated at 5.58–7.61%. In addition, the comparison of the T. lineatum assembly with 13 other Coleopteran genomes using QUAST ( Additional file 1 , Supplementary Table 2 ) confirmed a smaller overall assembly size and a distinct genomic profile with an N 0 value of 915 kb, which is lower than most other beetle genome assemblies ( Table 1, Figure 1a ). Additionally, the assembled genome of T. lineatum is smaller than the bark beetle species included in this comparison and exhibits moderate contiguity ( Table 1, Figure 1a, Additional file 1 , Supplementary Table 2 ). Telomeric repeats were not detected, which is likely due to technical limitations in assembling repetitive sequences. The compact genome structure may reflect the specialized fungus-feeding lifestyle of T. lineatum , consistent with previously observed patterns of streamlined genomes and reduced repetitive content in other insects, such as the Antarctic midge Belgica antarctica [59–61]. Genome annotation quality and iterative refinement Genome annotation involved both evidence-based and ab initio approaches, resulting in 19.92% of the genome being masked as repetitive DNA. Of this repetitive fraction, 14.82% comprised interspersed repeats, including 3.80% retroelements (primarily long terminal repeats at 3.69%), 2.71% DNA transposons, and 0.26% rolling-circle elements. Unclassified repeats accounted for 8.31%, simple repeats for 3.99%, and low-complexity regions for 0.79%. The low content of transposable elements (TEs) in the T. lineatum assembly contrasts with the higher percentages reported in other coleopteran species, such as D. ponderosae (21.12%), T. castaneum (28.90%), and D. valens (45.22%) [62]. The TEs are major contributors to genome expansion in eukaryotes, as they replicate and insert throughout the genome, thereby increasing the overall DNA content. Studies have shown a positive correlation between TE abundance and genome size, particularly in insects with small genomes. For example, the TE content ranges from less than 1% in the compact genome of the Antarctic midge ( Belgica antarctica ) to approximately 60% in the large genome of the migratory locust ( Locusta migratoria ) [63, 64]. The observed reduced proportion of TEs is consistent with the small genome size of T. lineatum and aligns with the positive correlation between genome size and TE abundance reported in other insects [59, 60]. While a high content of TEs has been proposed to enhance environmental adaptation and invasiveness in some insects by driving genomic evolution [64, 65], our findings suggest that a reduction in repetitive DNA may be an alternative strategy. However, assembly limitations, specifically moderate contiguity, suggest a small underestimation of the total repetitive content. The high unclassified fraction warrants improved assembly and specialized annotation in future studies. The iterative genome annotation pipeline using MAKER improved gene model quality across successive rounds, as evidenced by the progressive refinement of the AED distribution ( Additional File 2, Supplementary Figure 1b ), which can range from 0 (perfect agreement with evidence) to 1 [66]. The proportion of models with strong evidence support (AED ≤ 0.3) increased from 64.0% in Round 1 (R1) to 67.0% in Round 4 (R4), indicating a modest but consistent gain in annotation precision ( Additional File 1, Supplementary Table 3 ). Additionally, the completeness of the annotated genome was further assessed using BUSCO analysis based on the insecta_odb10 dataset (n = 1367). The analysis revealed that 95.2% of the expected genes were complete, with 93.1% identified as single-copy and 2.1% as duplicated. Only 1.9% and 2.9% of the BUSCOs were missing and fragmented, respectively, suggesting high completeness and quality of the T. lineatum genome annotation ( Additional File 1, Supplementary Table 4 ). Additionally, the genome annotation generated with MAKER identified 15,009 raw genes. After removing redundant sequences, this number was reduced to 14,830 unique gene predictions. From these, 11,551 (76.9%) were successfully assigned at least one functional domain or Gene Ontology (GO) term using InterPro and EggNOG ( Additional File 1, Supplementary Tables 5 and 6 ). The identified gene count (14,830) in T. lineatum is comparable to other coleopteran species, such as D. ponderosae (14,342) and Hycleus cichorii (13,813), and falls within the range observed in other coleopteran species [62, 67]. In addition, analysis of gene architecture ( Table 2 ) revealed an average of 6.33 exons per gene, exceeding most species in recent comparative studies (for example, D. virgifera with 4.51 and L. decemlineata with 5.06 exons), and closely matching C. maculatus (6.31) and S. oryzae (6.35) [67]. Notably, the T. lineatum genome is characterized by the compactness of its genes. The average exon length (211 bp) is shorter than in all other species included in our study, and the average intron length (322 bp) is an order of magnitude smaller than in species such as A. glabripennis (3,214 bp) and D. virgifera (10,348 bp) [67]. These findings are consistent with results from a recent study that reported unusually small introns in the manually annotated chemosensory genes of T. lineatum [35]. The combination of a high exon count and overall very short introns indicates a particularly compact gene architecture in T. lineatum , distinguishing it from other coleopterans, including other related scolytine species. This finding opens for further investigation of the evolutionary factors underlying such a genetic structure. Orthology inference and gene family analysis A total of 13,896 orthogroups were identified, encompassing 93.5% of the predicted genes across all ten species in the analysis ( Additional File 1, Supplementary Table 7 ) . In T. lineatum , 11,986 out of the 14,830 predicted genes (80.8%) were assigned to orthogroups, while 2,844 genes remained unassigned. Analysis of orthogroup overlap ( Additional File 1, Supplementary Table 8 ) revealed that T. lineatum shares the highest number of orthogroups with the scolytine species H. hampei (8,694 orthogroups), suggesting a closer relationship in terms of gene family content with this species. In contrast, T. lineatum shares the fewest orthogroups with the chrysomelid C. maculatus (3,716). Species-specific expansions were also identified: T. lineatum contained 78 species-specific orthogroups comprising 238 genes in total. This pattern suggests the presence of lineage-specific gene family expansions potentially related to the ecological specializations of T. lineatum . Comparative visualization using OrthoVenn3 further highlighted unique orthogroup distributions across selected species ( Figure 1B ). Gene family expansion and contraction in beetles have been proposed to be linked to ecological adaptations, environmental pressures, and evolutionary history [3, 36]. These changes often involve genes associated with metabolism, sensory systems, development, digestion, and detoxification processes [68, 69]. Across the analyzed beetle species, 33 gene families exhibited significant size changes (p ≤ 0.05) ( Additional File 1, Supplementary Table 9 ). In T. lineatum , 16 gene families were significantly expanded, while 17 were contracted. Notably, functional annotation of the contracted gene families revealed an enrichment of predicted domains related to key biological processes, including digestion, detoxification, host-environment interactions, and immunity. We found that the glycoside hydrolase family 28 (GH28, PF00295), which encodes plant cell wall degrading enzymes (PCWDEs), was significantly contracted in T. lineatum . The GH28 enzymes, especially polygalacturonases (PGs), are vital for pectin digestion, a major plant cell wall component, in many herbivorous beetles [70, 71]. These genes likely originated via horizontal gene transfer (HGT) from fungi or bacteria, then expanded through lineage-specific duplications, possibly to aid the exploitation of plant hosts. This expansion supports the evolution of herbivory in the Phytophaga clade [3, 72–75]. Prior studies have reported GH28 expansions in plant-feeding species such as D. virgifera , L. decemlineata , A. glabripennis , and I. typographus [31, 70, 76]. Also, our CAFE analysis confirmed such lineage-expansions in these species. In contrast, T. lineatum has a much smaller GH28 repertoire, consistent with its fungus-feeding lifestyle; even though the beetles bore inside the tree xylem they do not ingest this tissue directly [77], making these enzymes largely unnecessary. This contraction may be an example of how gene family changes can mirror ecological specializations, in this case a specialized diet. The evolution of the cytochrome P450 (PF00067) gene family is often closely associated with the ability of insects to adapt to chemically diverse environments, particularly in relation to detoxification mechanisms like host-plant interactions and insecticide resistance [78–80]. While expansions of this gene family are likely to enable insects to metabolize a broader range of foreign chemical compounds, our analysis showed that the P450 gene family is contracted in T. lineatum . Moreover, our results confirmed the expansion of this gene family in species such as the small hive beetle ( A. tumida ) and the coffee berry borer ( H. hampei ). The small hive beetle has a large P450 gene family with 116 genes, including notable expansions in the CYP3 and CYP4 clans, which may underlie its broad metabolic capacity and potential for insecticide resistance [81]. Similarly, the coffee berry borer has an extensive P450 gene repertoire that appears specialized for detoxifying unique defensive compounds present in its host, such as chlorogenic acid derivatives and caffeine [82]. This finding suggests that a large P450 family is not a universal response to environmental challenges, but rather a reflection of the specific selective pressures at play. The evolutionary forces shaping P450s are complex, with gene family size influenced by a combination of stochastic changes and natural selection [83, 84]. For instance, even among polyphagous beetles, the evidence for P450 expansions driven by dietary shifts is limited to a small number of orthologous groups, with other detoxification families showing more pronounced enrichment [85]. Thus, the contraction observed in T. lineatum may be a consequence of its specialized fungus-associated lifestyle and colonization of weakened or dying trees, which reduces exposure to diverse plant secondary metabolites, or may instead reflect reliance on alternative detoxification pathways. Symbiotic interactions are fundamental to insect survival, nutrition, development, and immunity [86–88]. T. lineatum exemplifies this, as its obligate mutualistic relationship with P. ferruginea is essential for larval development [22, 26, 89]. This association likely requires immune adaptations that enable the beetle to tolerate its fungal partner in the glandular mycangia (Joseph and Keyhani, 2021) while maintaining effective defenses against pathogens. Fungal symbionts are typically recognized by β-glucans, which activate pattern recognition receptors and initiate the Toll and Imd pathways, leading to NF-κB activation and the production of antimicrobial peptides [88]. In this context, the contraction of the serpin gene family (PF00079) in T. lineatum , with only a single retained copy, may represent an evolutionary adjustment of immune regulatory mechanisms. Serpins function as negative regulators of protease cascades in both the Toll and prophenoloxidase (PPO) pathways [90–92]. A reduced serpin repertoire could streamline immune regulation, potentially facilitating a stable relation with its obligate symbiont. Alternatively, this contraction may indicate a trade-off, reducing immune flexibility in favor of symbiont tolerance. Interestingly, a similar contraction of the serpin family was observed in the bark beetle I. typographus , whose associated fungi aid in successful spruce colonization, both by providing nutritional benefits to the beetle and by metabolizing host defense compounds [31, 93, 94] Similarly, the trypsin gene family (PF00089/IPR001254) was also contracted in T. lineatum . Although members of this family share the conserved trypsin-like catalytic domain (IPR001254), trypsins function as digestive serine proteases, hydrolyzing dietary proteins by cleaving peptide bonds at lysine and arginine residues, and also as key regulators of immunity and development [95]. Several trypsin-like proteases function as prophenoloxidase-activating factors (PPAFs), initiating the phenoloxidase cascade and melanization, a central defense mechanism against pathogens [96]. Thus, the contraction of this family in T. lineatum further supports the hypothesis that immune adaptability may be reduced in exchange for tolerance of its obligate fungal symbiont. In contrast, marked expansions in the trypsin gene family were identified in species such as D. virgifera , A. glabripennis , and A. tumida . This suggests that ecological context and symbiotic associations may play a crucial role in shaping the evolution of immune gene families across Coleoptera. The THAP domain (PF05485) family, which is involved in transcriptional and genomic regulation, was among the significantly expanded gene families in T. lineatum . The THAP domain is a type of DNA-binding domain characteristic of transcription factors, and its evolution is often linked to the activity of transposable elements (TEs), particularly the P-element superfamily [97, 98]. While the THAP domain family size varies across insects, notable lineage-specific expansions have been reported in species with high TE activity, such as the pea aphid ( Acyrthosiphon pisum ), which possesses hundreds of copies. This expansion is thought to be driven by the proliferation of TEs [99]. The significant expansion of the THAP domain family in the compact genome of T. lineatum , which shows a low overall content of repetitive DNA, presents an intriguing contrast. The expansion of a domain linked to TE proliferation suggests that the genome selectively retained the innovative products of a past evolutionary arms race [100, 101], keeping the beneficial TE-derived genes while actively shedding the non-functional repetitive content. This highlights an alternative evolutionary strategy: retaining the genetic innovation driven by TEs without keeping the genomic burden of the repetitive sequences. Further investigation is needed to determine the specific functions of these expanded THAP genes and their potential link to the unique genomic architecture of T. lineatum . We also found that the gene family CD80-like C2-set immunoglobulin (Ig) domain (PF08205) was significantly expanded in T. lineatum ( Figure 2 ) . In arthropods, proteins containing this C2-set Ig domain are fundamental for cell-cell recognition, adhesion, and immune signaling, acting as receptors or co-receptors that detect pathogens and activate defense pathways [102, 103]. The expansion of this family in T. lineatum suggests an evolutionary investment in the molecular mechanisms of immune recognition and communication. This is an interesting finding, given the obligate nutritional mutualism with P. ferruginea . Coexistence with a beneficial microorganism requires an immune system capable of distinguishing between symbionts and pathogens. We hypothesize that the expanded repertoire of CD80-like domains provides the molecular diversity necessary for immune discrimination, allowing the beetle to recognize and tolerate its fungal mutualist while maintaining the capacity to mount defenses against other invading microbes. Conclusions Our comparative genomic analysis of T. lineatum reveals distinct genomic signatures that likely reflect its specialized, obligate nutritional mutualism with its fungal symbiont, P. ferruginea . The results suggest that a small genome and significant changes in gene family size, particularly contractions, can be key components in the evolution of ecological specializations. The genome size, along with its reduced content of transposable elements (TEs) and its condensed gene architecture (short introns) differ from many other beetle species. While TE proliferation often drives genomic expansion and is linked to environmental adaptation, the genomic streamlining observed in T. lineatum may be a specialized adaptation to its fungus-cultivating niche. This contrasts with the genomic landscapes of polyphagous or herbivorous beetles that face a wider array of environmental and host-related challenges. The contraction of the GH28 family, which encodes plant cell wall-degrading enzymes, probably reflects the evolutionary shift from herbivory to fungivory. Similarly, the contraction of the cytochrome P450, serpin, and trypsin gene families suggests a potential trade-off between broad-spectrum metabolic and immune defense capabilities and the requirements of symbiotic tolerance. This reduction in immune-related gene families may represent a key mechanism enabling the beetle to maintain its relationship with its obligate fungal mutualist. In summary, this study sheds new light on the genomic basis of symbiosis in beetles. Our findings suggest that co-evolutionary relationships with microbial symbionts may lead to a reorganization of the insect genome, characterized not by the acquisition of new defenses but by the loss or reduction of gene families that may no longer be essential for survival. This genomic "pruning" or streamlining may reduce the metabolic cost of maintaining unnecessary physiological systems, while optimizing the insect for its specific, nutrient-rich niche. Abbreviations AED Annotation Edit Distance AMP Antimicrobial peptide CYP Cytochrome P450 GH28 Glycoside Hydrolase Family 28 HGT Horizontal gene transfer Ig Immunoglobulin Imd Immune deficiency pathway LTR Long terminal repeat NF-κB Nuclear factor kappa-B PCWDE Plant cell wall-degrading enzyme PG Polygalacturonase PPO Prophenoloxidase PPAF Prophenoloxidase-activating factor TE Transposable element THAP Thanatos-associated protein domain VOC Volatile organic compound Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding The study was funded by the Swedish Research Council FORMAS (grant #2018–01444 to M.N.A), the Max Planck Society (to H.V.), and the Max Planck Center next Generation Insect Chemical Ecology (nGICE, to C.L. and M.N.A). Author Contribution M.N.A. conceived the study and conceptualized it together with Z.M-O. Z.M-O. performed the genome annotation, bioinformatic analyses, molecular work, and drafted the manuscript, with M.N.A contributing to the initial draft. H.V. sequenced, assembled, and analyzed the quality of the *T. lineatum* genome. D.P. provided technical guidance in the bioinformatics analysis and assisted with interpretation. C.L. provided resources for the bioinformatics analysis. All authors provided scientific and editorial input to the manuscript and approved the final version for submission. Acknowledgements We thank Tomas Larsson and the Swedish Bioinformatics Advisory Program for advice on the bioinformatics analysis, and Twinkle Biswas for assistance with RNA isolations. We acknowledge support from the National Genomics Infrastructure in Genomics Production Stockholm, funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, and the Swedish Research Council. Additionally, computational resources were provided by the National Academic Infrastructure for Supercomputing in Sweden (NAISS) and the Swedish National Infrastructure for Computing (SNIC) at UPPMAX and Dardel PDC, partially funded by the Swedish Research Council through grant agreements no. 2022-23541and no. 2023–5461. Data Availability All datasets generated and/or analysed during the current study have been deposited in Figshare and are publicly available at the following repository: https://doi.org/10.6084/m9.figshare.30692345. The RNAseq reads have been deposited in the SRA database at NCBI under the accession number PRJNA1370798, and the whole Genome shotgun project has been deposited at GenBank under the accession JBSOPU000000000. In addition, all scripts, custom code, and a step-by-step reproducible workflow used for the comparative genomic analyses are openly available in the GitHub repository: https://github.com/lachemontes/comparativeGenomics_Tlin.git. References Stork NE. 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Transposable elements, contributors in the evolution of organisms (from an arms race to a source of raw materials). Heliyon. 2021;7:e06029. Tables Table 1. Comparative genome assembly statistics and annotation features for Trypodendron lineatum and nine additional coleopteran species. Features Tlin Ityp Dpon Hham Atum Agla Cmac Ldec Tcas Genome size (Mb) 83.62 236.81 223.59 162.57 259.96 706.95 1246.71 640.05 165.92 Number of contigs 833 272 2,110 8,198 8 9,866 938 21,825 2,062 Genome assembly quality Contig N50 915,233 6,654,004 16,553,750 340,248 36,783,356 678,234 9,445,077 139,401 15,265,516 Contig L50 21 12 4 89 3 269 38 1172 5 Complete BUSCO genes (%) 95.2 99.4 98.6 97.0 99.5 99.1 99.1 93.0 99.3 Genomic features G + C (%) 28.23 35.21 35.82 32.32 27.93 32.84 37.99 35.47 33.86 Gene annotation Number of genes 15,009 23,923 17,698 17,698 17,850 17,850 13,200 19,039 16,590 Species abbreviations: Tlin ( Trypodendron lineatum ), Ityp ( Ips typographus ), Dpon ( Dendroctonus ponderosae ), Hham ( Hypothenemus hampei ), Atum ( Aethina tumida ), Agla ( Anoplophora glabripennis ), Cmac ( Callosobruchus maculatus ), Ldec ( Leptinotarsa decemlineata ), and Tcas ( Tribolium castaneum ). Table 2. Statistics of the predicted gene models from Trypodendron lineatum (Tlin) and Ips typographus (Ityp) [31] . Statistic Tlin Ityp Number of genes 15,009 23,923 Total gene length (bp) 45,826,672 132,911,182 Longest gene (bp) 77,847 318,767 Average gene length (bp) 3,053 5,556 Average exon length (bp) 211 324 Average intron length (bp) 322 957 % of genome covered by genes 54.8 56 Average exons per gene 6.33 5 Average introns per gene 5.33 4 Number of genes containing Pfam domains 9,555 14,145 Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Supplementary Table 1.Summary of beetle species included in comparative genomics and orthology analyses. For each species, taxonomic family, GenBank accession number, protein sequence source, and inclusion status in ortholog analysis are indicated. Annotated proteins were downloaded in April 2025. Supplementary Table S2. Genome assembly statistics, including contig number, total assembly length, contig length metrics (N50, N90), and number of contigs greater than specific length thresholds for each beetle species used in the study. Supplementary Table S3. Cumulative distribution of Annotation Edit Distance (AED) scores across iterative MAKER rounds for Trypodendron lineatum . Supplementary Table S4. BUSCO assessment of genome annotation completeness for Trypodendron lineatum and eight additional Coleoptera species using the insecta_odb10 dataset. Supplementary Table S5. Functional annotation of Trypodendron lineatum gene models using InterProScan. Supplementary Table S6. Functional annotation of Trypodendron lineatum gene models using EggNOGmapper. Supplementary Table S7. Statistics summarizing the ortholog group analysis. Supplementary Table S8. Summary of orthogroup assignment statistics for Trypodendron lineatum and related beetle species. Supplementary Table S9. Results of gene family expansion and contraction analysis in Trypodendron lineatum and nine additional species using CAFE. AdditionalFile2.pdf Supplementary Figure 1. Genome annotation workflow and quality improvement across iterative MAKER. rounds for Trypodendron lineatum . Supplementary Figure 2A. Phylogenetic analysis of orthogroup OG0000151 annotated as Glycosyl hydrolases family 28 (PF00295). Supplementary Figure 2B. Phylogenetic analysis of orthogroup OG0000036 annotated as Cytochrome P450 (PF00067). Supplementary Figure 2C. Phylogenetic analysis of orthogroup OG0000140 annotated as Serpin (PF00079). Supplementary Figure 2D. Phylogenetic analysis of orthogroup OG0000044 annotated as Trypsin (PF00089). Supplementary Figure 2E. Phylogenetic analysis of orthogroup OG0000079 annotated as THAP domain (PF05485). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8230855","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560334684,"identity":"e3324dff-c253-4b76-b7a9-101e6f9e780c","order_by":0,"name":"Zaide Montes-Ortiz","email":"data:image/png;base64,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","orcid":"","institution":"Lund University","correspondingAuthor":true,"prefix":"","firstName":"Zaide","middleName":"","lastName":"Montes-Ortiz","suffix":""},{"id":560334685,"identity":"f8a92df5-706a-4cc2-9ee0-7ea23ae45577","order_by":1,"name":"Daniel Powell","email":"","orcid":"","institution":"University of the Sunshine Coast","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Powell","suffix":""},{"id":560334686,"identity":"e4b36141-b64c-4897-b64c-0c49f0bc15e7","order_by":2,"name":"Heiko Vogel","email":"","orcid":"","institution":"Max Planck Institute for Chemical Ecology","correspondingAuthor":false,"prefix":"","firstName":"Heiko","middleName":"","lastName":"Vogel","suffix":""},{"id":560334687,"identity":"11545681-76e9-4205-adbd-149f14968a7a","order_by":3,"name":"Christer Löfstedt","email":"","orcid":"","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Christer","middleName":"","lastName":"Löfstedt","suffix":""},{"id":560334688,"identity":"e6c7cf1c-9db1-45e9-9995-24582243c249","order_by":4,"name":"Martin N. Andersson","email":"","orcid":"","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"N.","lastName":"Andersson","suffix":""}],"badges":[],"createdAt":"2025-11-28 13:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8230855/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8230855/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98301688,"identity":"3e1a907b-53d1-4620-89a5-0be2f08a37ad","added_by":"auto","created_at":"2025-12-16 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10:12:03","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226901,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/7ea2d5308dc66b3b58336002.html"},{"id":98301681,"identity":"dca24990-8b67-4331-830a-0633fd8e21b7","added_by":"auto","created_at":"2025-12-16 10:12:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genomic features and gene family evolution across beetle species.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Cumulative genome assembly length (in Mbp) plotted against contig index for \u003cem\u003eTrypodendron lineatum\u003c/em\u003e and 9 other coleopteran species, illustrating variation in assembly contiguity and size. The species included are \u003cem\u003eAethina tumida\u003c/em\u003e(Atum), \u003cem\u003eAnoplophora glabripennis \u003c/em\u003e(Agla), \u003cem\u003eCallosobruchus maculatus\u003c/em\u003e(Cmac), \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e (Dpon), \u003cem\u003eDiabrotica virgifera \u003c/em\u003e(Dvir), \u003cem\u003eHypothenemus hampei\u003c/em\u003e (Hham), \u003cem\u003eIps typographus\u003c/em\u003e (Ityp), and \u003cem\u003eLeptinotarsa decemlineata \u003c/em\u003eLdec). \u003cstrong\u003eB)\u003c/strong\u003e Venn diagram of orthologous groups shared between \u003cem\u003eT. lineatum\u003c/em\u003e (striped ambrosia beetle), \u003cem\u003eI.\u003c/em\u003e \u003cem\u003etypographus\u003c/em\u003e, \u003cem\u003eD. ponderosae\u003c/em\u003e (two bark beetles), and \u003cem\u003eA. glabripennis\u003c/em\u003e (a polyphagous cerambycid wood-borer). \u003cstrong\u003eC)\u003c/strong\u003e Gene family expansions and contractions inferred using CAFE for each species. Numbers indicate the total count of expanded (red) and contracted (black) gene families. Species are grouped by family. The stacked bar plots on the right represent the distribution of different types of genes in corresponding species, including single-copy, unique, and clustered genes.\u003c/p\u003e","description":"","filename":"floatimage122.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/a4a04d23e65a1dbbfebf31aa.jpeg"},{"id":98436586,"identity":"e658a96b-e382-47f8-87ab-7290eced9b63","added_by":"auto","created_at":"2025-12-17 16:55:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1414198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of orthogroup OG0000184 annotated as CD80-like C2-set immunoglobulin domain (PF08205).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic tree of the ortholog group OG0000184, included 77 protein sequences from ten beetle species: \u003cem\u003eTrypodendron lineatum\u003c/em\u003e (Tlin), \u003cem\u003eIps typographus\u003c/em\u003e(Ityp), \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e (Dpon), \u003cem\u003eHypothenemus hampei\u003c/em\u003e(Hham), \u003cem\u003eAnoplophora glabripennis\u003c/em\u003e (Agla), \u003cem\u003eCallosobruchus maculatus \u003c/em\u003e(Cmac), \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e (Ldec), \u003cem\u003eDiabrotica virgifera\u003c/em\u003e (Dvir), \u003cem\u003eAethina tumida\u003c/em\u003e (Atum) and \u003cem\u003eTribolium castaneum\u003c/em\u003e (Tcas). Support values are labelled next to the branches, which were derived from 100 bootstrap replicates. This gene family was significantly expanded in \u003cem\u003eT. lineatum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/ee75f2db5d9cbb134d7dfdd8.jpeg"},{"id":98445617,"identity":"bc498cc4-1475-4a31-8a9b-ea95375b0bb9","added_by":"auto","created_at":"2025-12-17 17:20:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2914833,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/3c93208c-c4e9-4b73-8ab9-a49cef404583.pdf"},{"id":98301693,"identity":"34d435b1-db63-4bd5-8da3-c9094786c592","added_by":"auto","created_at":"2025-12-16 10:12:03","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11439048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1.\u003c/strong\u003eSummary of beetle species included in comparative genomics and orthology analyses. For each species, taxonomic family, GenBank accession number, protein sequence source, and inclusion status in ortholog analysis are indicated. Annotated proteins were downloaded in April 2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S2. \u003c/strong\u003eGenome assembly statistics, including contig number, total assembly length, contig length metrics (N50, N90), and number of contigs greater than specific length thresholds for each beetle species used in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S3. \u003c/strong\u003eCumulative distribution of Annotation Edit Distance (AED) scores across iterative MAKER rounds for \u003cem\u003eTrypodendron lineatum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S4. \u003c/strong\u003eBUSCO assessment of genome annotation completeness for \u003cem\u003eTrypodendron lineatum \u003c/em\u003eand eight additional Coleoptera species using the insecta_odb10 dataset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S5. \u003c/strong\u003eFunctional annotation of \u003cem\u003eTrypodendron lineatum\u003c/em\u003e gene models using InterProScan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S6. \u003c/strong\u003eFunctional annotation of \u003cem\u003eTrypodendron lineatum\u003c/em\u003e gene models using EggNOGmapper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S7. \u003c/strong\u003eStatistics summarizing the ortholog group analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S8. \u003c/strong\u003eSummary of orthogroup assignment statistics for \u003cem\u003eTrypodendron lineatum \u003c/em\u003eand related beetle species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table S9. \u003c/strong\u003eResults of gene family expansion and contraction analysis in \u003cem\u003eTrypodendron lineatum \u003c/em\u003eand nine additional species using CAFE.\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/795e17b29a82390fec9b2cee.xlsx"},{"id":98434690,"identity":"65ce6c41-9577-4070-87f1-0d4a24fa90ad","added_by":"auto","created_at":"2025-12-17 16:52:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":762858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u003c/strong\u003eGenome annotation workflow and quality improvement across iterative MAKER. rounds for \u003cem\u003eTrypodendron lineatum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2A. \u003c/strong\u003ePhylogenetic analysis of orthogroup OG0000151 annotated as Glycosyl hydrolases family 28 (PF00295).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2B. \u003c/strong\u003ePhylogenetic analysis of orthogroup OG0000036 annotated as Cytochrome P450 (PF00067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2C. \u003c/strong\u003ePhylogenetic analysis of orthogroup OG0000140 annotated as Serpin (PF00079).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2D. \u003c/strong\u003ePhylogenetic analysis of orthogroup OG0000044 annotated as Trypsin (PF00089).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2E. \u003c/strong\u003ePhylogenetic analysis of orthogroup OG0000079 annotated as THAP domain (PF05485).\u003c/p\u003e","description":"","filename":"AdditionalFile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8230855/v1/7914ddf1a988b1c2c0c4f09b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative genomics reveal signatures of ecological specialization in the striped ambrosia beetle Trypodendron lineatum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBeetles (Coleoptera) represent one of the most species-rich and ecologically diverse lineages in the animal kingdom, comprising over 380,000 described species across a wide range of terrestrial and freshwater ecosystems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, their evolutionary success has been attributed in part to their remarkable diversity of feeding strategies, allowing them to exploit a wide variety of ecological niches [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These include phytophagy (feeding on plants), saproxyly (feeding on decaying wood) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], predation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and mycophagy (fungus-feeding) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Importantly, several of these feeding strategies are often accompanied by symbiotic relationships with microbes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This dietary versatility has not only fostered extensive diversification but has also driven functional innovations at morphological (e.g., specialized mouthparts) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], physiological (e.g., digestive enzymes) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and genomic (e.g., detoxification-related and chemosensory genes) levels [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBark- and ambrosia beetles (family Curculionidae) are both members of the subfamily Scolytinae. However, ambrosia beetles are a polyphyletic group with members also in Platypodinae, and they differ from bark beetles in terms of feeding behavior, larval development, and associations with symbiotic microbes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Bark beetles primarily feed on the host tree phloem inoculated with beetle-associated symbiotic fungi. Inside the phloem, their larvae construct individual tunnels away from the maternal gallery, with limited parent-offspring interaction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In contrast, ambrosia beetles bore into the xylem and cultivate obligate mutualistic fungi within their galleries. Rather than feeding on wood directly, they rely on these fungal gardens as their sole nutritional source [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This tight interdependence exemplifies a co-evolved obligate nutritional mutualism that has shaped the ecological strategies and life histories of ambrosia beetles in fundamentally different ways from their bark beetle relatives [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This symbiotic relationship is maintained through complex mechanisms, including the ability of ambrosia beetles to store and maintain fungal spores in a dormant state within specialized glandular mycangia. This process has likely had a significant evolutionary impact on their immune system and associated genes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe striped ambrosia beetle, \u003cem\u003eTrypodendron lineatum\u003c/em\u003e Olivier, is a significant pest species of Holarctic conifer forests [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It targets stressed or dying conifer trees, boring into the xylem to establish larval galleries, which are inoculated with spores of its obligate fungal mutualist, \u003cem\u003ePhialophoropsis ferruginea\u003c/em\u003e (Mathiesen-K\u0026auml;\u0026auml;rik) (Ascomycota) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This fungus-farming lifestyle is a prime example of niche construction, enabling \u003cem\u003eT. lineatum\u003c/em\u003e to modify its environment effectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo find suitable host trees for colonization, volatile organic compounds from decaying conifers, such as α-pinene and ethanol [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], attract \u003cem\u003eT. lineatum\u003c/em\u003e, whereas volatiles from non-host angiosperm trees are avoided [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].The beetle also uses the female-produced aggregation pheromone (+)-lineatin (3,3,7-trimethyl-2,9-dioxatricyclononane) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], which attracts both sexes to trees. Moreover, the presence of olfactory sensory neurons that specifically respond to volatiles produced by its fungal mutualist highlights a finely tuned sensory system for locating suitable habitats and maintaining the fungal association [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Hence, \u003cem\u003eT. lineatum\u003c/em\u003e could be considered to exhibit a narrow ecological niche and strong dependence on both host trees and fungal partners, which makes it an ideal model for studying the genomic consequences of mutualistic specialization, especially in comparison with closely related bark beetle species with different ecological adaptations [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Whereas large-scale genomic annotation and analyses of the bark beetles \u003cem\u003eIps typographus\u003c/em\u003e L. (Eurasian spruce bark beetle) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e Hopkins (mountain pine beetle) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and \u003cem\u003eHypothenemus hampei\u003c/em\u003e Ferarri (coffee berry borer) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] have yielded insights into these species\u0026rsquo; ecological specializations, similar analysis has, to the best of our knowledge, never been performed on an ambrosia beetle. In fact, \u003cem\u003eT. lineatum\u003c/em\u003e is one of the few ambrosia beetle species with a sequenced genome [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], making this species a prime target for such analysis.\u003c/p\u003e \u003cp\u003eEcological specialization, particularly in species that engage in obligate mutualisms, is often accompanied by genomic signatures that reflect adaptation to narrow niches [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A previous study showed that \u003cem\u003eT. lineatum\u003c/em\u003e possesses a reduced repertoire of chemoreceptor-coding genes compared to other scolytine beetles, potentially reflecting a streamlined sensory system tailored to its specific associations with its host trees and fungal mutualist [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, it remains unclear whether such specialization has had broader impacts on other gene families involved in \u003cem\u003ee.g.\u003c/em\u003e digestion, detoxification, immunity, or development. To address this, we performed a comparative genomic and orthology analysis of \u003cem\u003eT. lineatum\u003c/em\u003e and nine other beetle species, including related scolytine bark beetles, as well as other less related species. We focused on the expansion and contraction of gene families as a proxy for functional adaptation, with the aim of uncovering how ecological constraints and mutualistic strategies have shaped the genome of \u003cem\u003eT. lineatum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData sources\u003c/h2\u003e \u003cp\u003eThe genome sequencing (EMBL-EBI accession number PRJEB74033; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/view/PRJEB74033\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/ena/browser/view/PRJEB74033\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and assembly were performed previously (see [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] for details), which involved extracting genomic DNA from \u003cem\u003eT. lineatum\u003c/em\u003e of Swedish origin and sequencing with Oxford Nanopore (~\u0026thinsp;190 x genome coverage) and Illumina platforms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This comprehensive approach resulted in a high-quality assembly, divided between 833 contigs (N50: 915 kbp; longest contig: 4.16 Mbp) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] that formed the basis for the present work. Additionally, three distinct datasets were used in this study: (1) a set of target beetle genomes, (2) annotated proteins retrieved from public databases, and (3) whole-body transcriptomes of \u003cem\u003eT. lineatum\u003c/em\u003e generated in the present study as well as an antennal transcriptome from [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (NCBI accession number PRJNA1126204; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/PRJNA1126204\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra/PRJNA1126204\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In brief, for (1), we downloaded 13 beetle genomes from different families (January 2024) deposited in the public database \u003cem\u003eGenBank\u003c/em\u003e from the National Center for Biotechnology Information (NCBI); accession numbers for each species can be found in \u003cb\u003eAdditional file 1, Supplementary Table\u0026nbsp;1\u003c/b\u003e. For (2), we downloaded a non-redundant, well-annotated set of proteins from the \u003cem\u003eRefSeq\u003c/em\u003e by using the query \u0026ldquo;(\"beetle species\"[Organism] OR beetle specie [All Fields]) AND refseq[filter],\u0026rdquo; where \u0026ldquo;beetle species\u0026rdquo; was replaced by each of the ten species used for the orthology analysis. The data from \u003cem\u003eI. typographus\u003c/em\u003e, \u003cem\u003eH. hampei\u003c/em\u003e, and \u003cem\u003eCallosobruchus maculatus\u003c/em\u003e Fabricius (Chrysomelidae) were obtained from public repositories (\u003cb\u003eAdditional file 1, Supplementary Table\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome size estimation and identification of telomeres\u003c/h3\u003e\n\u003cp\u003eThe genome size of \u003cem\u003eT. lineatum\u003c/em\u003e was estimated using a k-mer-based approach. Raw sequencing reads were first processed with Trimmomatic (v0.36) for quality control [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. K-mer frequency analysis was then performed using Jellyfish (v2.2.10) with a k-mer length of 21, and the resulting distribution was analyzed by GenomeScope (v2.0) to determine genome size, heterozygosity, and repeat content [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To contextualize the estimated genome size, we used QUAST (v.5.3.0) to compare it with the genome assemblies of 14 other beetle species [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, telomeric regions within the \u003cem\u003eT. lineatum\u003c/em\u003e genome were searched for using the Telomere Identification toolKit (tidk) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and the FindTelomer.py script (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/JanaSperschneider/FindTelomeres\u003c/span\u003e\u003cspan address=\"https://github.com/JanaSperschneider/FindTelomeres\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We specifically searched for the telomeric repeat motifs TTAGG, TTAGGG, CCCTAA, and TCAGG commonly found in Coleoptera [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTranscriptome sequencing, read mapping, and assembly\u003c/h3\u003e\n\u003cp\u003eAdult \u003cem\u003eT. lineatum\u003c/em\u003e males and females for transcriptome sequencing were collected from a conifer dominated forest in T\u0026aring;gar\u0026ouml;d, South Sweden, during May 2023 using pheromone-baited (Lineatin Kombi dispensers, Witasek, Austria) traps (WitaTrap, 12 funnel size, Witasek). The whole bodies of six females and six males (in two separate samples) were used to isolate total RNA using the RNeasy Minikit (Qiagen, Hilden, Germany), according to the manufacturer\u0026rsquo;s instructions. Then, the RNA was DNAse-treated and subjected to library construction using the Illumina TrueSeq stranded mRNA (polyA) kit (Illumina, San Diego, CA, USA). The samples were sequenced on a NovaSeq6000 (NovaSeq Control Software 1.8.1/RTA v3.4.4) with a 151nt(Read1)-19nt(Index1)-10nt(Index2)-151nt(Read2) setup using 'NovaSeqXp' workflow in 'S4' mode flowcell. The raw reads were initially assessed for quality using FastQC (v2.2.1). Adapter trimming and quality filtering were performed using Trimmomatic [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], followed by a secondary quality assessment with FastQC (v2.2.1) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], with summary reports generated by MultiQC [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. High-quality reads were mapped to the \u003cem\u003eT. lineatum\u003c/em\u003e reference genome using HISAT2 (v2.2.1) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eDe novo\u003c/em\u003e transcriptome assembly was conducted with Trinity (v2.9.1) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Additionally, CD-HIT-EST was performed with a sequence identity threshold of 0.98 to reduce redundancy among the assembled transcripts [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Finally, coding regions within the assembled transcripts were predicted using TransDecoder (v5.7.0) (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/TransDecoder/TransDecoder\u003c/span\u003e\u003cspan address=\"https://github.com/TransDecoder/TransDecoder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGenome annotation and quality assessment\u003c/h3\u003e\n\u003cp\u003eThe genome of \u003cem\u003eT. lineatum\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was annotated using two complementary pipelines: MAKER (v3.01.2) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and BRAKER (v3) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Before annotation, repetitive elements were identified by constructing an \u003cem\u003ead hoc\u003c/em\u003e repeat library directly from the \u003cem\u003eT. lineatum\u003c/em\u003e genome assembly using RepeatModeler (v4.1.0) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This custom library was combined with the comprehensive Repbase repeat database and subsequently applied with RepeatMasker (v4.0.8) to soft-mask repetitive regions in the genome [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Following the masking step, the MAKER annotation pipeline was executed in four iterative rounds. In the initial round, the annotation process incorporated various sources of evidence data, including transcriptome assemblies derived from whole-body and antennal tissues of \u003cem\u003eT. lineatum\u003c/em\u003e, as well as the predicted protein sets from nine coleopteran species (\u003cem\u003eD. ponderosae\u003c/em\u003e, \u003cem\u003eDendroctonus valens\u003c/em\u003e LeConte, \u003cem\u003eH. hampei\u003c/em\u003e, \u003cem\u003eI. typographus\u003c/em\u003e (all Curculionidae), \u003cem\u003eAnoplophora glabripennis\u003c/em\u003e Motschulsky (Cerambycidae), \u003cem\u003eDiabrotica virgifera\u003c/em\u003e LeConte, \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e Say (both Chrysomelidae), \u003cem\u003eNicrophorus vespilloides\u003c/em\u003e Herbst (Silphidae), \u003cem\u003eTribolium castaneum\u003c/em\u003e Herbst (Tenebrionidae)) and \u003cem\u003eDrosophila melanogaster\u003c/em\u003e Meigen (Diptera) (\u003cb\u003eAdditional file 1, Supplementary Table\u0026nbsp;1\u003c/b\u003e). Additionally, the Insecta_Odb10 database (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://busco.ezlab.org/\u003c/span\u003e\u003cspan address=\"https://busco.ezlab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), containing 1,367 core genes representing 75 insect species across 14 orders, was included to enhance the accuracy and completeness of the first gene model prediction.\u003c/p\u003e \u003cp\u003eIn the second round, the gene models generated in the initial round were used as input to train gene prediction software, specifically AUGUSTUS with BUSCO and SNAP. The outputs from these trained predictors were then integrated into MAKER to generate refined second-round gene models, progressively improving annotation quality. Consequently, for the third round, the gene models from the second round served as input, resulting in a new generation of gene models. Finally, a fourth round of annotation was performed by integrating gene predictions generated by the BRAKER3 (v3). These BRAKER3-generated gene models were merged with the MAKER annotation to produce a comprehensive and robust set of final gene annotations. After each round, the Annotation Edit Distance (AED) distribution was calculated. Finally, the redundancy and duplicated sequences were removed from the genome annotation generated with CD-HIT with an identity threshold of 98% [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and the assembly quality and completeness assessment were performed using BUSCO with the arthropoda_Odb10 database [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. All scripts, parameter files, and detailed commands used in the genome annotation workflow are publicly available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/lachemontes/comparativeGenomics_Tlin\u003c/span\u003e\u003cspan address=\"https://github.com/lachemontes/comparativeGenomics_Tlin\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eFunctional annotation and orthologs gene detection\u003c/h3\u003e\n\u003cp\u003eFor functional annotation, InterProScan (v2.1.4-2) was used to assign functional categories to the set of predicted protein-coding genes [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The analysis included the following databases: Gene3D, ProSite, Patterns, PANTHER, CDD, Pfam, Phobius, SUPERFAMILY, and TMHMM. In addition, eggNOG-mapper v1.0.3 was used to assign Gene Ontology (GO) terms based on the eggNOG database [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Then, before ortholog detection, a data pre-processing step was performed to improve sequence quality and consistency. Redundant protein sequences were clustered with CD-HIT at a 98% identity threshold, and sequences shorter than 100 amino acids were removed.\u003c/p\u003e \u003cp\u003eSubsequently, orthology inference was performed using OrthoFinder (v2.5.2) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Protein sequences from ten beetle species (\u003cem\u003eT. lineatum\u003c/em\u003e, \u003cem\u003eI. typographus\u003c/em\u003e, \u003cem\u003eD. ponderosae\u003c/em\u003e, \u003cem\u003eH. hampei\u003c/em\u003e, \u003cem\u003eT. castaneum\u003c/em\u003e, \u003cem\u003eA. glabripennis\u003c/em\u003e, \u003cem\u003eC. maculatus\u003c/em\u003e, \u003cem\u003eL. decemlineata\u003c/em\u003e, \u003cem\u003eD. virgifera\u003c/em\u003e, and \u003cem\u003eAethina tumida\u003c/em\u003e Murray (Nitidulidae)) were included. OrthoFinder was run using gene tree inference (-M msa), multiple sequence alignment (-A mafft), and the default tree inference program. Finally, to assign putative functions to orthogroups, InterProScan (v2.1.4-2) and BLAST searches (e-value\u0026thinsp;\u0026lt;\u0026thinsp;1e\u0026thinsp;\u0026minus;\u0026thinsp;5) against the NCBI non-redundant insect database (accessed April 2025) were performed, retaining only non-redundant and significant hits. This functional annotation of orthogroups was critical for subsequent gene family evolution analyses with CAFE, allowing biological interpretation of significantly expanded and contracted gene families [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene family evolution: expansions and contractions\u003c/h2\u003e \u003cp\u003eWe used CAFE (v5) with the 13,896 orthogroups identified during the OrthoFinder analysis to investigate the evolutionary dynamics of gene families. Gene family count data from these orthogroups were then reformatted to be compatible with CAFE requirements. In this analysis, we were able to estimate gene turnover rates (λ) for the 10 above-mentioned beetle species from five taxonomic families, to infer ancestral gene counts for each species, and to estimate gene gain/loss rates for each lineage of the phylogeny.\u003c/p\u003e \u003cp\u003eTo improve phylogenetic inference beyond the default tree construction method (FastTree) used by OrthoFinder, we used the \u0026ldquo;SpeciesTreeAlignment\u0026rdquo; output to reconstruct a species tree with IQ-TREE [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The best-fit evolutionary model was selected automatically using ModelFinder [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and was evaluated through 1,000 bootstrap iterations. The resulting phylogenetic tree was converted into an ultrametric tree using the \u0026lsquo;ape\u0026rsquo; package (v3.0) in R. Divergence time estimates for \u003cem\u003eT. castaneum\u003c/em\u003e and \u003cem\u003eI. typographus\u003c/em\u003e were obtained from TimeTree5 to calibrate the ultrametric tree (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://timetree.org\u003c/span\u003e\u003cspan address=\"https://timetree.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReconstruction of phylogenetic trees\u003c/h3\u003e\n\u003cp\u003eFor each gene family identified as significantly contracted or expanded, we reconstructed unrooted phylogenetic trees using the approximate maximum-likelihood method implemented in IQ-TREE (v2.4.0) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The resulting phylogenetic trees (\u003cb\u003eAdditional File 2, Supplementary Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-E\u003c/b\u003e) were then visualized and annotated using iTOL (v.7) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eGenome size and telomeric regions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe k-mer analysis of raw sequencing reads produced an estimated genome size for \u003cem\u003eT. lineatum\u003c/em\u003e ranging from 74.44 Mb to 83.62 Mb, which is smaller than typically observed in other beetle species (\u003cstrong\u003e\u003cem\u003eTable 1\u003c/em\u003e\u003c/strong\u003e). Heterozygosity was estimated at 5.58\u0026ndash;7.61%. In addition, the comparison of the \u003cem\u003eT. lineatum\u003c/em\u003e assembly with 13 other Coleopteran genomes using QUAST (\u003cstrong\u003e\u003cem\u003eAdditional file 1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e, Supplementary Table 2\u003c/em\u003e\u003c/strong\u003e) confirmed a smaller overall assembly size and a distinct genomic profile with an N\u003csub\u003e0\u003c/sub\u003e value of 915 kb, which is lower than most other beetle genome assemblies (\u003cstrong\u003e\u003cem\u003eTable 1, Figure 1a\u003c/em\u003e\u003c/strong\u003e). Additionally, the assembled genome of \u003cem\u003eT. lineatum\u003c/em\u003e is smaller than the bark beetle species included in this comparison and exhibits moderate contiguity (\u003cstrong\u003e\u003cem\u003eTable 1, Figure 1a, Additional file 1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e, Supplementary Table 2\u003c/em\u003e\u003c/strong\u003e). Telomeric repeats were not detected, which is likely due to technical limitations in assembling repetitive sequences. The compact genome structure may reflect the specialized fungus-feeding lifestyle of \u003cem\u003eT. lineatum\u003c/em\u003e, consistent with previously observed patterns of streamlined genomes and reduced repetitive content in other insects, such as the Antarctic midge \u003cem\u003eBelgica antarctica\u003c/em\u003e [59\u0026ndash;61].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome annotation quality and iterative refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome annotation involved both evidence-based and \u003cem\u003eab initio\u003c/em\u003e approaches, resulting in 19.92% of the genome being masked as repetitive DNA. Of this repetitive fraction, 14.82% comprised interspersed repeats, including 3.80% retroelements (primarily long terminal repeats at 3.69%), 2.71% DNA transposons, and 0.26% rolling-circle elements. Unclassified repeats accounted for 8.31%, simple repeats for 3.99%, and low-complexity regions for 0.79%. The low content of transposable elements (TEs) in the \u003cem\u003eT. lineatum\u003c/em\u003e assembly contrasts with the higher percentages reported in other coleopteran species, such as \u003cem\u003eD. ponderosae\u003c/em\u003e (21.12%), \u003cem\u003eT. castaneum\u003c/em\u003e (28.90%), and \u003cem\u003eD. valens\u003c/em\u003e (45.22%) [62]. The TEs are major contributors to genome expansion in eukaryotes, as they replicate and insert throughout the genome, thereby increasing the overall DNA content. Studies have shown a positive correlation between TE abundance and genome size, particularly in insects with small genomes. For example, the TE content ranges from less than 1% in the compact genome of the Antarctic midge (\u003cem\u003eBelgica antarctica\u003c/em\u003e) to approximately 60% in the large genome of the migratory locust (\u003cem\u003eLocusta migratoria\u003c/em\u003e) [63, 64]. The observed reduced proportion of TEs is consistent with the small genome size of \u003cem\u003eT. lineatum\u003c/em\u003e and aligns with the positive correlation between genome size and TE abundance reported in other insects [59, 60]. While a high content of TEs has been proposed to enhance environmental adaptation and invasiveness in some insects by driving genomic evolution [64, 65], our findings suggest that a reduction in repetitive DNA may be an alternative strategy. However, assembly limitations, specifically moderate contiguity, suggest a small underestimation of the total repetitive content. The high unclassified fraction warrants improved assembly and specialized annotation in future studies.\u003c/p\u003e\n\u003cp\u003eThe iterative genome annotation pipeline using MAKER improved gene model quality across successive rounds, as evidenced by the progressive refinement of the AED distribution (\u003cstrong\u003e\u003cem\u003eAdditional File 2, Supplementary Figure 1b\u003c/em\u003e\u003c/strong\u003e), which can range from 0 (perfect agreement with evidence) to 1 [66]. The proportion of models with strong evidence support (AED \u0026le; 0.3) increased from 64.0% in Round 1 (R1) to 67.0% in Round 4 (R4), indicating a modest but consistent gain in annotation precision (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Table 3\u003c/em\u003e\u003c/strong\u003e). Additionally, the completeness of the annotated genome was further assessed using BUSCO analysis based on the insecta_odb10 dataset (n = 1367). The analysis revealed that 95.2% of the expected genes were complete, with 93.1% identified as single-copy and 2.1% as duplicated. Only 1.9% and 2.9% of the BUSCOs were missing and fragmented, respectively, suggesting high completeness and quality of the \u003cem\u003eT. lineatum\u003c/em\u003e genome annotation (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Table 4\u003c/em\u003e\u003c/strong\u003e). Additionally, the genome annotation generated with MAKER identified 15,009 raw genes. After removing redundant sequences, this number was reduced to 14,830 unique gene predictions. From these, 11,551 (76.9%) were successfully assigned at least one functional domain or Gene Ontology (GO) term using InterPro and EggNOG (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Tables 5 and 6\u003c/em\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe identified gene count (14,830) in \u003cem\u003eT. lineatum\u0026nbsp;\u003c/em\u003eis comparable to other coleopteran species, such as \u003cem\u003eD. ponderosae\u003c/em\u003e (14,342) and \u003cem\u003eHycleus cichorii\u003c/em\u003e (13,813), and falls within the range observed in other coleopteran species [62, 67]. In addition, analysis of gene architecture (\u003cstrong\u003eTable 2\u003c/strong\u003e) revealed an average of 6.33 exons per gene, exceeding most species in recent comparative studies (for example, \u003cem\u003eD. virgifera\u003c/em\u003e with 4.51 and \u003cem\u003eL. decemlineata\u003c/em\u003e with 5.06 exons), and closely matching \u003cem\u003eC. maculatus\u003c/em\u003e (6.31) and \u003cem\u003eS. oryzae\u003c/em\u003e (6.35) [67]. Notably, the \u003cem\u003eT. lineatum\u003c/em\u003e genome is characterized by the compactness of its genes. The average exon length (211 bp) is shorter than in all other species included in our study, and the average intron length (322 bp) is an order of magnitude smaller than in species such as \u003cem\u003eA. glabripennis\u003c/em\u003e (3,214 bp) and \u003cem\u003eD. virgifera\u003c/em\u003e (10,348 bp) [67]. These findings are consistent with results from a recent study that reported unusually small introns in the manually annotated chemosensory genes of \u003cem\u003eT. lineatum\u003c/em\u003e [35]. The combination of a high exon count and overall very short introns indicates a particularly compact gene architecture in\u003cem\u003e\u0026nbsp;T. lineatum\u003c/em\u003e, distinguishing it from other coleopterans, including other related scolytine species. This finding opens for further investigation of the evolutionary factors underlying such a genetic structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrthology inference and gene family analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 13,896 orthogroups were identified, encompassing 93.5% of the predicted genes across all ten species in the analysis (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Table 7\u003c/em\u003e\u003c/strong\u003e)\u003cem\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003eIn \u003cem\u003eT. lineatum\u003c/em\u003e, 11,986 out of the 14,830 predicted genes (80.8%) were assigned to orthogroups, while 2,844 genes remained unassigned. Analysis of orthogroup overlap (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Table 8\u003c/em\u003e\u003c/strong\u003e) revealed that \u003cem\u003eT. lineatum\u003c/em\u003e shares the highest number of orthogroups with the scolytine species \u003cem\u003eH. hampei\u003c/em\u003e (8,694 orthogroups), suggesting a closer relationship in terms of gene family content with this species. In contrast, \u003cem\u003eT. lineatum\u003c/em\u003e shares the fewest orthogroups with the chrysomelid \u003cem\u003eC. maculatus\u003c/em\u003e (3,716). Species-specific expansions were also identified: \u003cem\u003eT. lineatum\u003c/em\u003e contained 78 species-specific orthogroups comprising 238 genes in total. This pattern suggests the presence of lineage-specific gene family expansions potentially related to the ecological specializations of \u003cem\u003eT. lineatum\u003c/em\u003e. Comparative visualization using OrthoVenn3 further highlighted unique orthogroup distributions across selected species (\u003cstrong\u003e\u003cem\u003eFigure 1B\u003c/em\u003e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGene family expansion and contraction in beetles have been proposed to be linked to ecological adaptations, environmental pressures, and evolutionary history [3, 36]. These changes often involve genes associated with metabolism, sensory systems, development, digestion, and detoxification processes\u0026nbsp;[68, 69]. Across the analyzed beetle species, 33 gene families exhibited significant size changes (p \u0026le; 0.05) (\u003cstrong\u003e\u003cem\u003eAdditional File 1, Supplementary Table 9\u003c/em\u003e\u003c/strong\u003e). In \u003cem\u003eT. lineatum\u003c/em\u003e, 16 gene families were significantly expanded, while 17 were contracted. Notably, functional annotation of the contracted gene families revealed an enrichment of predicted domains related to key biological processes, including digestion, detoxification, host-environment interactions, and immunity.\u003c/p\u003e\n\u003cp\u003eWe found that the glycoside hydrolase family 28 (GH28, PF00295), which encodes plant cell wall degrading enzymes (PCWDEs), was significantly contracted in \u003cem\u003eT. lineatum\u003c/em\u003e. The GH28 enzymes, especially polygalacturonases (PGs), are vital for pectin digestion, a major plant cell wall component, in many herbivorous beetles [70, 71]. These genes likely originated via horizontal gene transfer (HGT) from fungi or bacteria, then expanded through lineage-specific duplications, possibly to aid the exploitation of plant hosts. This expansion supports the evolution of herbivory in the Phytophaga clade [3, 72\u0026ndash;75]. Prior studies have reported GH28 expansions in plant-feeding species such as \u003cem\u003eD. virgifera\u003c/em\u003e, \u003cem\u003eL. decemlineata\u003c/em\u003e, \u003cem\u003eA. glabripennis\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;I. typographus\u003c/em\u003e [31, 70, 76]. Also, our CAFE analysis confirmed such lineage-expansions in these species. In contrast, \u003cem\u003eT. lineatum\u003c/em\u003e has a much smaller GH28 repertoire, consistent with its fungus-feeding lifestyle; even though the beetles bore inside the tree xylem they do not ingest this tissue directly [77], making these enzymes largely unnecessary. This contraction may be an example of how gene family changes can mirror ecological specializations, in this case a specialized diet.\u003c/p\u003e\n\u003cp\u003eThe evolution of the cytochrome P450 (PF00067) gene family is often closely associated with the ability of insects to adapt to chemically diverse environments, particularly in relation to detoxification mechanisms like host-plant interactions and insecticide resistance [78\u0026ndash;80]. While expansions of this gene family are likely to enable insects to metabolize a broader range of foreign chemical compounds, our analysis showed that the P450 gene family is contracted in \u003cem\u003eT. lineatum\u003c/em\u003e. Moreover, our results confirmed the expansion of this gene family in species such as the small hive beetle (\u003cem\u003eA. tumida\u003c/em\u003e) and the coffee berry borer (\u003cem\u003eH. hampei\u003c/em\u003e). The small hive beetle has a large P450 gene family with 116 genes, including notable expansions in the CYP3 and CYP4 clans, which may underlie its broad metabolic capacity and potential for insecticide resistance [81]. Similarly, the coffee berry borer has an extensive P450 gene repertoire that appears specialized for detoxifying unique defensive compounds present in its host, such as chlorogenic acid derivatives and caffeine [82]. This finding suggests that a large P450 family is not a universal response to environmental challenges, but rather a reflection of the specific selective pressures at play. The evolutionary forces shaping P450s are complex, with gene family size influenced by a combination of stochastic changes and natural selection [83, 84]. For instance, even among polyphagous beetles, the evidence for P450 expansions driven by dietary shifts is limited to a small number of orthologous groups, with other detoxification families showing more pronounced enrichment [85]. Thus, the contraction observed in \u003cem\u003eT. lineatum\u003c/em\u003e may be a consequence of its specialized fungus-associated lifestyle and colonization of weakened or dying trees, which reduces exposure to diverse plant secondary metabolites, or may instead reflect reliance on alternative detoxification pathways.\u003c/p\u003e\n\u003cp\u003eSymbiotic interactions are fundamental to insect survival, nutrition, development, and immunity [86\u0026ndash;88]. \u003cem\u003eT. lineatum\u003c/em\u003e exemplifies this, as its obligate mutualistic relationship with \u003cem\u003eP. ferruginea\u003c/em\u003e is essential for larval development [22, 26, 89]. This association likely requires immune adaptations that enable the beetle to tolerate its fungal partner in the glandular mycangia (Joseph and Keyhani, 2021) while maintaining effective defenses against pathogens. Fungal symbionts are typically recognized by \u0026beta;-glucans, which activate pattern recognition receptors and initiate the Toll and Imd pathways, leading to NF-\u0026kappa;B activation and the production of antimicrobial peptides [88]. In this context, the contraction of the serpin gene family (PF00079) in \u003cem\u003eT. lineatum\u003c/em\u003e, with only a single retained copy, may represent an evolutionary adjustment of immune regulatory mechanisms. Serpins function as negative regulators of protease cascades in both the Toll and prophenoloxidase (PPO) pathways [90\u0026ndash;92]. \u0026nbsp;A reduced serpin repertoire could streamline immune regulation, potentially facilitating a stable relation with its obligate symbiont. Alternatively, this contraction may indicate a trade-off, reducing immune flexibility in favor of symbiont tolerance. Interestingly, a similar contraction of the serpin family was observed in the bark beetle \u003cem\u003eI. typographus\u003c/em\u003e, whose associated fungi aid in successful spruce colonization, both by providing nutritional benefits to the beetle and by metabolizing host defense compounds [31, 93, 94]\u003c/p\u003e\n\u003cp\u003eSimilarly, the trypsin gene family (PF00089/IPR001254) was also contracted in \u003cem\u003eT. lineatum\u003c/em\u003e. Although members of this family share the conserved trypsin-like catalytic domain (IPR001254), trypsins function as digestive serine proteases, hydrolyzing dietary proteins by cleaving peptide bonds at lysine and arginine residues, and also as key regulators of immunity and development [95]. Several trypsin-like proteases function as prophenoloxidase-activating factors (PPAFs), initiating the phenoloxidase cascade and melanization, a central defense mechanism against pathogens [96]. Thus, the contraction of this family in \u003cem\u003eT. lineatum\u003c/em\u003e further supports the hypothesis that immune adaptability may be reduced in exchange for tolerance of its obligate fungal symbiont. In contrast, marked expansions in the trypsin gene family were identified in species such as \u003cem\u003eD. virgifera\u003c/em\u003e, \u003cem\u003eA. glabripennis\u003c/em\u003e, and \u003cem\u003eA. tumida\u003c/em\u003e. This suggests that ecological context and symbiotic associations may play a crucial role in shaping the evolution of immune gene families across Coleoptera.\u003c/p\u003e\n\u003cp\u003eThe THAP domain (PF05485) family, which is involved in transcriptional and genomic regulation, was among the significantly expanded gene families in \u003cem\u003eT. lineatum\u003c/em\u003e. The THAP domain is a type of DNA-binding domain characteristic of transcription factors, and its evolution is often linked to the activity of transposable elements (TEs), particularly the P-element superfamily [97, 98]. While the THAP domain family size varies across insects, notable lineage-specific expansions have been reported in species with high TE activity, such as the pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e), which possesses hundreds of copies. This expansion is thought to be driven by the proliferation of TEs [99]. The significant expansion of the THAP domain family in the compact genome of \u003cem\u003eT. lineatum\u003c/em\u003e, which shows a low overall content of repetitive DNA, presents an intriguing contrast. The expansion of a domain linked to TE proliferation suggests that the genome selectively retained the innovative products of a past evolutionary arms race [100, 101], keeping the beneficial TE-derived genes while actively shedding the non-functional repetitive content. This highlights an alternative evolutionary strategy: retaining the genetic innovation driven by TEs without keeping the genomic burden of the repetitive sequences. Further investigation is needed to determine the specific functions of these expanded THAP genes and their potential link to the unique genomic architecture of \u003cem\u003eT. lineatum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe also found that the gene family CD80-like C2-set immunoglobulin (Ig) domain (PF08205) was significantly expanded in \u003cem\u003eT. lineatum (\u003cstrong\u003eFigure 2\u003c/strong\u003e)\u003c/em\u003e. In arthropods, proteins containing this C2-set Ig domain are fundamental for cell-cell recognition, adhesion, and immune signaling, acting as receptors or co-receptors that detect pathogens and activate defense pathways [102, 103]. The expansion of this family in \u003cem\u003eT. lineatum\u003c/em\u003e suggests an evolutionary investment in the molecular mechanisms of immune recognition and communication. This is an interesting finding, given the obligate nutritional mutualism with \u003cem\u003eP. ferruginea\u003c/em\u003e. Coexistence with a beneficial microorganism requires an immune system capable of distinguishing between symbionts and pathogens. We hypothesize that the expanded repertoire of CD80-like domains provides the molecular diversity necessary for immune discrimination, allowing the beetle to recognize and tolerate its fungal mutualist while maintaining the capacity to mount defenses against other invading microbes.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur comparative genomic analysis of \u003cem\u003eT. lineatum\u003c/em\u003e reveals distinct genomic signatures that likely reflect its specialized, obligate nutritional mutualism with its fungal symbiont, \u003cem\u003eP. ferruginea\u003c/em\u003e. The results suggest that a small genome and significant changes in gene family size, particularly contractions, can be key components in the evolution of ecological specializations. The genome size, along with its reduced content of transposable elements (TEs) and its condensed gene architecture (short introns) differ from many other beetle species. While TE proliferation often drives genomic expansion and is linked to environmental adaptation, the genomic streamlining observed in \u003cem\u003eT. lineatum\u003c/em\u003e may be a specialized adaptation to its fungus-cultivating niche. This contrasts with the genomic landscapes of polyphagous or herbivorous beetles that face a wider array of environmental and host-related challenges. The contraction of the GH28 family, which encodes plant cell wall-degrading enzymes, probably reflects the evolutionary shift from herbivory to fungivory. Similarly, the contraction of the cytochrome P450, serpin, and trypsin gene families suggests a potential trade-off between broad-spectrum metabolic and immune defense capabilities and the requirements of symbiotic tolerance. This reduction in immune-related gene families may represent a key mechanism enabling the beetle to maintain its relationship with its obligate fungal mutualist.\u003c/p\u003e \u003cp\u003eIn summary, this study sheds new light on the genomic basis of symbiosis in beetles. Our findings suggest that co-evolutionary relationships with microbial symbionts may lead to a reorganization of the insect genome, characterized not by the acquisition of new defenses but by the loss or reduction of gene families that may no longer be essential for survival. This genomic \"pruning\" or streamlining may reduce the metabolic cost of maintaining unnecessary physiological systems, while optimizing the insect for its specific, nutrient-rich niche.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAED\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnnotation Edit Distance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial peptide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCYP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome P450\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGH28\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlycoside Hydrolase Family 28\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorizontal gene transfer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoglobulin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eImd\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmune deficiency pathway\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLong terminal repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor kappa-B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCWDE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlant cell wall-degrading enzyme\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolygalacturonase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProphenoloxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProphenoloxidase-activating factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransposable element\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTHAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThanatos-associated protein domain\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVolatile organic compound\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was funded by the Swedish Research Council FORMAS (grant #2018\u0026ndash;01444 to M.N.A), the Max Planck Society (to H.V.), and the Max Planck Center next Generation Insect Chemical Ecology (nGICE, to C.L. and M.N.A).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.N.A. conceived the study and conceptualized it together with Z.M-O. Z.M-O. performed the genome annotation, bioinformatic analyses, molecular work, and drafted the manuscript, with M.N.A contributing to the initial draft. H.V. sequenced, assembled, and analyzed the quality of the *T. lineatum* genome. D.P. provided technical guidance in the bioinformatics analysis and assisted with interpretation. C.L. provided resources for the bioinformatics analysis. All authors provided scientific and editorial input to the manuscript and approved the final version for submission.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Tomas Larsson and the Swedish Bioinformatics Advisory Program for advice on the bioinformatics analysis, and Twinkle Biswas for assistance with RNA isolations. We acknowledge support from the National Genomics Infrastructure in Genomics Production Stockholm, funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, and the Swedish Research Council. Additionally, computational resources were provided by the National Academic Infrastructure for Supercomputing in Sweden (NAISS) and the Swedish National Infrastructure for Computing (SNIC) at UPPMAX and Dardel PDC, partially funded by the Swedish Research Council through grant agreements no. 2022-23541and no. 2023\u0026ndash;5461.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll datasets generated and/or analysed during the current study have been deposited in Figshare and are publicly available at the following repository: https://doi.org/10.6084/m9.figshare.30692345. The RNAseq reads have been deposited in the SRA database at NCBI under the accession number PRJNA1370798, and the whole Genome shotgun project has been deposited at GenBank under the accession JBSOPU000000000. In addition, all scripts, custom code, and a step-by-step reproducible workflow used for the comparative genomic analyses are openly available in the GitHub repository: https://github.com/lachemontes/comparativeGenomics_Tlin.git.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStork NE. How many species of insects and other terrestrial arthropods are there on Earth? Annu Rev Entomol. 2018;63:31\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S-Q, Che L-H, Li Y, Liang D, Pang H, Ślipiński A, et al. Evolutionary history of Coleoptera revealed by extensive sampling of genes and species. Nat Commun. 2018;9:205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKenna DD, Shin S, Ahrens D, Balke M, Beza-Beza C, Clarke DJ, et al. The evolution and genomic basis of beetle diversity. 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The ambrosia symbiosis: from evolutionary ecology to practical management. Annu Rev Entomol. 2017;62:285\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu N, Li M, Gong Y, Liu F, Li T. Cytochrome P450s: their expression, regulation, and role in insecticide resistance. Pestic Biochem Physiol. 2015;120:77\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu K, Song Y, Zeng R. The role of cytochrome P450-mediated detoxification in insect adaptation to xenobiotics. Curr Opin Insect Sci. 2021;43:103\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang T, Li T, Feng X, Li M, Liu S, Liu N. Multiple cytochrome P450 genes confer high levels of permethrin resistance in mosquitoes (\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e). Sci Rep. 2021;11:9041.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans JD, McKenna D, Scully E, Cook SC, Dainat B, Egekwu N, et al. Genome of the small hive beetle (\u003cem\u003eAethina tumida\u003c/em\u003e, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory. GigaScience. 2018;7:giy138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVega FE, Brown SM, Chen H, Shen E, Nair MB, Ceja-Navarro JA, et al. Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, \u003cem\u003eHypothenemus hampei\u003c/em\u003e. Sci Rep. 2015;5:12525.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGood RT, Gramzow L, Battlay P, Sztal T, Batterham P, Robin C. The molecular evolution of cytochrome P450 genes within and between \u003cem\u003eDrosophila\u003c/em\u003e species. Genome Biol Evol. 2014;6:1118\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSezutsu H, Le Goff G, Feyereisen R. Origins of P450 diversity. Philos Trans R Soc Lond B Biol Sci. 2013;368:20120428.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeppey M, Ioannidis P, Emerson BC, Pitteloud C, Robinson-Rechavi M, Roux J, et al. Genomic signatures accompanying the dietary shift to phytophagy in polyphagan beetles. Genome Biol. 2019;20:98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerasategui A, Shukla S, Salem H, Kaltenpoth M. Potential applications of insect symbionts in biotechnology. Appl Microbiol Biotechnol. 2016;100:1567\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCornwallis CK, Van \u0026rsquo;t Padje A, Ellers J, Klein M, Jackson R, Kiers ET, et al. Symbioses shape feeding niches and diversification across insects. Nat Ecol Evol. 2023;7:1022\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePonton F, Tan YX, Forster CC, Austin AJ, English S, Cotter SC, et al. The complex interactions between nutrition, immunity and infection in insects. J Exp Biol. 2023;226:jeb245714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayers CG, Harrington TC, McNew DL, Roeper RA, Biedermann PHW, Masuya H, et al. Four mycangium types and four genera of ambrosia fungi suggest a complex history of fungus farming in the ambrosia beetle tribe Xyloterini. Mycologia. 2020;112:1104\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanost MR, Jiang H. Clip-domain serine proteases as immune factors in insect hemolymph. Curr Opin Insect Sci. 2015;11:47\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeekins DA, Kanost MR, Michel K. Serpins in arthropod biology. Semin Cell Dev Biol. 2017;62:105\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShakeel M, Xu X, De Mandal S, Jin F. Role of serine protease inhibitors in insect\u0026ndash;host\u0026ndash;pathogen interactions. Arch Insect Biochem Physiol. 2019;102:e21556.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNetherer S, Kandasamy D, Jirosov\u0026aacute; A, Kalinov\u0026aacute; B, Schebeck M, Schlyter F. Interactions among Norway spruce, the bark beetle \u003cem\u003eIps typographus\u003c/em\u003e and its fungal symbionts in times of drought. J Pest Sci. 2021;94:591\u0026ndash;614.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao T, Kandasamy D, Krokene P, Chen J, Gershenzon J, Hammerbacher A. Fungal associates of the tree-killing bark beetle \u003cem\u003eIps typographus\u003c/em\u003e vary in virulence, ability to degrade conifer phenolics and influence bark beetle tunneling behavior. Fungal Ecol. 2019;38:71\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuhlia-Almaz\u0026aacute;n A, S\u0026aacute;nchez-Paz A, Garc\u0026iacute;a-Carre\u0026ntilde;o FL. Invertebrate trypsins: a review. J Comp Physiol B. 2008;178:655\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZdybicka-Barabas A, Stączek S, Kunat-Budzyńska M, Cytryńska M. Innate immunity in insects: the lights and shadows of phenoloxidase system activation. Int J Mol Sci. 2025;26:1320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Z, Qin J, Zhou X, Zhang Y. Insect transcription factors: a landscape of their structures and biological functions in \u003cem\u003eDrosophila\u003c/em\u003e and beyond. Int J Mol Sci. 2018;19:3691.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabogal A, Lyubimov AY, Corn JE, Berger JM, Rio DC. THAP proteins target specific DNA sites through bipartite recognition of adjacent major and minor grooves. Nat Struct Mol Biol. 2010;17:117\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVidal NM, Grazziotin AL, Iyer LM, Aravind L, Venancio TM. Transcription factors, chromatin proteins and the diversification of Hemiptera. Insect Biochem Mol Biol. 2016;69:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJangam D, Feschotte C, Betr\u0026aacute;n E. Transposable element domestication as an adaptation to evolutionary conflicts. Trends Genet. 2017;33:817\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePourrajab F, Hekmatimoghaddam S. Transposable elements, contributors in the evolution of organisms (from an arms race to a source of raw materials). Heliyon. 2021;7:e06029.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"1001\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"bottom\" style=\"width: 89.9101%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eComparative genome assembly statistics and annotation features for \u003cem\u003eTrypodendron lineatum\u0026nbsp;\u003c/em\u003eand nine additional coleopteran species.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeatures\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTlin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eItyp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDpon\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHham\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAtum\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgla\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCmac\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLdec\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTcas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eGenome size (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e83.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e236.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e223.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e162.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e259.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e706.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e1246.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e640.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e165.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eNumber of contigs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e272\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e2,110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e8,198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e9,866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e21,825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e2,062\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenome assembly quality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eContig N50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e915,233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e6,654,004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e16,553,750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e340,248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e36,783,356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e678,234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e9,445,077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e139,401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e15,265,516\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eContig L50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e1172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eComplete BUSCO genes (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e95.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e99.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e98.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e97.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e99.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e99.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e99.1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e93.0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e99.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic features\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eG + C (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e28.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e35.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e35.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e32.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e27.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e32.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e37.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e35.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e33.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene annotation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003eNumber of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e15,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e23,923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e17,698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e17,698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e17,850\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e17,850\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e13,200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e19,039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e16,590\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 19.4805%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7.99201%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.09091%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 8.09191%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.0899%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSpecies abbreviations: Tlin (\u003cem\u003eTrypodendron lineatum\u003c/em\u003e), Ityp (\u003cem\u003eIps typographus\u003c/em\u003e), Dpon (\u003cem\u003eDendroctonus ponderosae\u003c/em\u003e), Hham (\u003cem\u003eHypothenemus hampei\u003c/em\u003e), Atum (\u003cem\u003eAethina tumida\u003c/em\u003e), Agla (\u003cem\u003eAnoplophora glabripennis\u003c/em\u003e), Cmac (\u003cem\u003eCallosobruchus maculatus\u003c/em\u003e), Ldec (\u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e), and Tcas (\u003cem\u003eTribolium castaneum\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"bottom\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eStatistics of the predicted gene models from \u003cem\u003eTrypodendron lineatum\u0026nbsp;\u003c/em\u003e(Tlin) and \u003cem\u003eIps typographus\u0026nbsp;\u003c/em\u003e(Ityp) [31] .\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTlin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eItyp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eNumber of genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e15,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e23,923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eTotal gene length (bp) \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e45,826,672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e132,911,182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eLongest gene (bp) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e77,847\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e318,767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eAverage gene length (bp) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e3,053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e5,556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eAverage exon length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eAverage intron length (bp) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003e% of genome covered by genes \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e54.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eAverage exons per gene \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e6.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eAverage introns per gene \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e5.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003eNumber of genes containing Pfam domains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e9,555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e14,145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63.0332%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 17.5355%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 18.4834%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 0.473934%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Coleoptera, Curculionidae, Scolytinae, Symbiosis, Genome annotation, Gene family evolution, Immune gene contraction, Detoxification","lastPublishedDoi":"10.21203/rs.3.rs-8230855/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8230855/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBeetles (Coleoptera) exhibit remarkable dietary versatility, which may drive genomic innovations. Ambrosia beetles (Curculionidae: Scolytinae) have evolved specific feeding habits and intricate relationships with symbiotic fungi. The striped ambrosia beetle \u003cem\u003eTrypodendron lineatum\u003c/em\u003e is a pest of conifers, relying on its obligate nutritional mutualist \u003cem\u003ePhialophoropsis ferruginea\u003c/em\u003e for survival. The beetles cultivate the fungi inside their galleries in the tree\u0026rsquo;s xylem, with the fungi serving as their sole food source. We hypothesize that this lifestyle is associated with genomic signatures that may reflect important adaptations. Hence, we performed a comparative genomic analysis between \u003cem\u003eT. lineatum\u003c/em\u003e and nine other beetle species, including related scolytine bark beetles, to uncover genomic signatures of this specialization, focusing on gene families involved in e.g. digestion, detoxification, and immunity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe small genome of \u003cem\u003eT. lineatum\u003c/em\u003e (74.4\u0026ndash;83.6 Mb) exhibits comparatively low levels of repetitive DNA (19.9%), including a reduced proportion of transposable elements, and unusually short introns. Annotation generated 14,830 high-quality gene predictions, most of which were supported by transcript evidence or functional domains. Comparative orthology analysis identified 13,896 orthogroups, with \u003cem\u003eT. lineatum\u003c/em\u003e showing 78 species-specific orthogroups and a set of gene family changes which may reflect its ecological specializations. Thirty-three \u003cem\u003eT. lineatum\u003c/em\u003e gene families showed significant size changes, including 16 expansions and 17 contractions. Notably, gene families associated with digestion, detoxification, and immunity were contracted. These included glycoside hydrolase 28, cytochrome P450, serpin, and trypsin families, suggesting reduced reliance on plant-based digestion and broad-spectrum immune defenses. In contrast, expansions in the THAP domain and CD80-like immunoglobulin domain families indicate selective retention and diversification of genes involved in genomic regulation and immune recognition.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results suggests that the genome of \u003cem\u003eT. lineatum\u003c/em\u003e is streamlined, characterized by a low repeat content and compact gene architecture. The observed contractions in key gene families involved in plant digestion, detoxification, and immunity likely represent genomic signatures of its obligate mutualistic specialization and narrow ecological niche. Our findings provide the first insights into the genomic adaptations of fungus-farming ambrosia beetles, suggesting that co-evolved insect-microbe mutualisms may lead to reductions in a variety of insect gene families.\u003c/p\u003e","manuscriptTitle":"Comparative genomics reveal signatures of ecological specialization in the striped ambrosia beetle Trypodendron lineatum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 10:11:58","doi":"10.21203/rs.3.rs-8230855/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-08T06:10:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T18:08:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T02:45:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-30T06:11:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17409091151315574086651054760258452110","date":"2025-12-17T19:51:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-14T16:41:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253940710602088048244794194526117843525","date":"2025-12-13T05:11:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247110321305339699847683574214383220907","date":"2025-12-12T06:18:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110039994042674664579188261174268211646","date":"2025-12-11T11:30:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195754212945789778037520105777217230480","date":"2025-12-11T02:57:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-11T02:51:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T00:07:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T00:07:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-11-28T13:22:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"335e6c0a-aaac-4fa4-bd6b-439b597067f3","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T08:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 10:11:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8230855","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8230855","identity":"rs-8230855","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}