A chromosomal-level genome assembly of Promethis valgipes Marseul, 1876 (Coleoptera: Tenebrionidae)

Abstract

Promethis valgipes is an important fungus-eating beetle distributed in central China, Korea, and Japan. However, research on its biology and evolution has been limited by the lack of a high-quality genome. Here, we report a chromosome-level genome assembly of P. valgipes, generated using an integrated approach that combines PacBio HiFi long reads, Illumina short reads, and Hi-C data. The final genome assembly spans 213.69 Mb, with 98.91% (211.35 Mb) of the total sequence anchored to 10 chromosomes. The genome assembly consists of 19 scaffolds and 30 contigs, with scaffold and contig N50 values of 21.50 Mb and 17.10 Mb, respectively. BUSCO analysis using the insecta_odb10 dataset (n = 1,367) indicated high completeness, with 99.7% of conserved genes detected (99.3% single-copy and 0.4% duplicated). Repetitive elements constituted 41.74% (89.19 Mb) of the genome, and 13544 protein-coding genes were identified. This high-quality genome provides a foundation for studying the ecology, genetics, and evolution of P. valgipes.

Background & Summary

The Tenebrionidae (Coleoptera) is a large and diverse beetle family, comprising over 30,000 described species across eight to ten subfamilies^1,2. Members of this family are widely distributed globally and exhibit remarkable adaptability to diverse environmental conditions³. Most tenebrionid species are detritivorous at both larval and adult stages, playing essential roles in terrestrial food webs⁴. Based on ecological preferences, Tenebrionidae can be broadly divided into two groups: species closely associated with trees, and those whose larvae transition from decaying wood to soil habitats⁴. The latter group is particularly well-adapted to arid environments and is widely distributed in desert ecosystems. These beetles have evolved a suite of morphological, physiological, and behavioral adaptations that facilitate survival under extreme aridity, thereby contributing substantially to nutrient cycling in desert ecosystems^5,6.

The diversity of feeding strategies and habitats within Tenebrionidae has driven considerable morphological variation and substantial economic impacts. Many species are notable agricultural pests; for instance, Tribolium spp. are cosmopolitan pests of stored products such as flour, corn, and peanuts⁷. Conversely, the protein-rich larvae of these species have potential as sustainable feed for pets, including reptiles and birds⁸. Beyond economic relevance, certain tenebrionid species also provide ecological benefits. Notably, Tenebrio molitor Linnaeus and Uloma spp. can biodegrade polystyrene through synergistic interactions with their gut microbiota⁹. High-quality genome assemblies are essential for understanding the biology, ecology, and evolutionary history of Tenebrionidae. As a major stored-product pest and a representative of the largest insect order, Tribolium castaneum is a well-established model for insect development¹⁰. Its genome shows expansions in chemoreceptor and detoxification gene families, and its development is more insect-typical than that of Drosophila, making it a valuable system for functional genomics and RNAi-based gene characterization¹¹. More recently, chromosome-level genome assemblies have been published for eight Tenebrionidae beetles, including T. castaneum¹², T. molitor¹³, Ulomoides dermestoides¹⁴, Prionychus ater¹⁵, Eledona agricola¹⁶, Lagria hirta¹⁷, Diaperis boleti¹⁸, and Phaleria cadaverina¹⁹ providing important genomic resources for comparative and evolutionary studies within the family. Despite recent progress, high-quality genomes in Tenebrionidae remain limited, and additional chromosome-level assemblies are needed to advance comparative genomics and clarify the evolutionary history of this diverse group.

In this study, we present a chromosome-level genome assembly of Promethis valgipes (Marseul, 1876), an important fungus-feeding beetle distributed across central China, Korea, and Japan²⁰. P. valgipes causes substantial damage to shiitake mushroom cultivation at both larval and adult stages. The larvae bore into cultivation logs, leading to wood decay and rendering them unsuitable for production, while adults feed on fruiting bodies, resulting in significant yield losses. To date, no high-quality genomic resource has been reported for the subfamily Stenochiinae. Therefore, the high-quality genome of P. valgipes generated in this study provides an important reference for this subfamily and will contribute to a deeper understanding of its evolutionary biology. The assembly was generated using PacBio HiFi, Illumina, and Hi-C sequencing, followed by comprehensive annotation of repetitive elements, non-coding RNAs, and protein-coding genes. This high-quality reference genome provides a valuable resource for investigating the evolutionary history, ecological adaptations, and functional genomics of Tenebrionidae.

Methods

Sample collection and sequencing

For parallel DNA and RNA sequencing, an adult male of P. valgipes was collected in Xinyang, Henan Province, China, on 6 July 2025. Muscle tissue, specifically from the pronotum and posterior abdomen, was dissected from the specimen and used for DNA/RNA extraction and Hi-C library construction. To minimize exogenous contamination, the dissected tissue was rinsed in phosphate-buffered saline for 10 minutes. The sample was then flash-frozen in liquid nitrogen for 20 minutes and stored at −80 °C until sequencing.

Genomic DNA was extracted using the CTAB method and then purified with a Grandomics Genomic DNA Kit for downstream sequencing²¹. Total RNA was isolated from the same individual using TRIzol Reagent (Thermo Fisher Scientific). A PCR-free Illumina library was subsequently constructed from the extracted DNA using the TruSeq DNA PCR-Free Kit, generating 150 bp paired-end reads. Hi-C libraries were prepared following a standard workflow that included formaldehyde cross-linking and MboI digestion²². Short-read sequencing was conducted on the Illumina NovaSeq. 6000 platform, while long-read sequencing was performed on the PacBio Sequel II (HiFi mode). For the PacBio platform, a 20 kb SMRTbell library was generated using the SMRTbell Express Template Prep Kit 2.0. All library preparation and sequencing were performed by Berry Genomics (Beijing, China). In total, the sequencing effort generated 83.2 Gb of data, comprising 19.81 Gb of PacBio HiFi long reads (~92.71 × coverage), 30.91 Gb of Illumina short reads (144.67×), 21.22 Gb of Hi-C data (99.30×), and 11.26 Gb of RNA-seq data (Table 1).

Table 1.

Statistics of the sequencing data used for genome assembly.

Libraries	Insert sizes (bp)	Clean data (Gb)	Sequencing coverage (×)
Illumina	350	30.91	144.67
PacBio HiFi	20 Kb	19.81	92.71
Hi-C	350	21.22	99.30
RNA	350	11.26	—

Genome assembly

To guide the selection of appropriate assembly strategies and parameter settings, we conducted a genome survey to estimate genome size, heterozygosity, and repeat content. Illumina sequencing reads were quality-controlled using BBTools v38.82²³. The processing included duplicate removal with clumpify.sh, adapter and low-quality base trimming (Q < 20) using bbduk.sh, and filtering out reads containing more than five ambiguous bases. Poly-A, poly-G, and poly-C tails longer than 10 bp were also trimmed. The filtered reads were then analyzed using GenomeScope v2.0²⁴ for genome survey estimation. The estimated genome size was 209.69 Mb, with duplicated sequences accounting for 31.37% of the genome. The heterozygosity rate was 1.51%, indicating relatively high genetic diversity (Fig. 1).

Fig. 1.

Genome size, heterozygosity, and repeat content of the Promethis valgipes genome were estimated using GenomeScope. The genome was estimated to be 209.69 Mb in size, with a heterozygosity rate of 1.51% and a duplication rate of 31.37%.

The draft genome of P. valgipes was assembled from PacBio HiFi reads using Hifiasm v0.19.8²⁵ with default parameters. To reduce redundancy due to heterozygosity, haplotigs were removed using Purge_Dups v1.2.5²⁶ with a haploid cutoff of 70. Hi-C reads were mapped to the contigs using Juicer v1.6.2²⁷, and the assembly was subsequently scaffolded into chromosome-scale linkage groups with 3D-DNA v180922²⁸. We manually inspected the assembly using Juicebox v1.11.0²⁷ and corrected potential misassemblies to generate the final Hi-C–based assembly. Potential contaminants were identified using MMseqs. 2 v11.12²⁹ to perform BLASTN-like searches against the NCBI nucleotide and UniVec databases. In addition, a dedicated BLASTN³⁰ (BLAST + v2.11.0) search against UniVec was conducted to detect vector sequences. Sequences showing > 90% similarity to known contaminants were automatically removed using stringent filtering criteria, whereas sequences with 80–90% similarity were further validated by BLASTN searches against the NCBI nucleotide database. The final assembled genome passed NCBI’s automatic contamination screening with no significant contaminant sequences detected, reflecting high sample quality and sequencing accuracy.

To distinguish autosomes from sex chromosomes, PacBio HiFi and Illumina reads were mapped to the final assembly, and chromosome-level coverage was calculated. However, repeat-rich chromosomes may exhibit artificially elevated coverage due to ambiguous read mapping. Chr02, Chr04, and Chr09 showed approximately two-fold higher coverage than other chromosomes, likely due to their higher repeat content rather than true copy-number variation. Therefore, chromosome-specific coverage values were removed from Table 3 to avoid misinterpretation. To distinguish autosomes from sex chromosomes, the X chromosome was further validated through synteny analysis with Tribolium castaneum, taking advantage of the conserved characteristics of insect X chromosomes. Syntenic blocks were identified using MCScanX³¹ and visualized with TBtools³². Overall, chromosome 10 was identified as the X chromosome based on its strong syntenic correspondence with the X chromosome of T. castaneum (Fig. 3). Scaffolds longer than 10 kb in the P. valgipes genome were retained for downstream assembly evaluation and genome annotation. The chromosome-level assembly spanned 213.69 Mb with a GC content of 36.97% (Table 2). The assembly achieved scaffold and contig N50 values of 21.50 Mb and 17.10 Mb, respectively, and comprised 19 scaffolds and 30 contigs. Notably, 98.91% (211.35 Mb) of the assembly was anchored to 10 chromosomes, with chromosome sizes ranging from 11.38 Mb to 36.93 Mb (Fig. 2, Fig. 4; Table 3). Furthermore, the final assembly closely matches the genome size estimated by k-mer analysis using GenomeScope (209.69 Mb).

Table 3.

Statistics for chromosomes sequence length.

Chromosome ID	Sequences Length (bp)
Chr01	36925200
Chr02	24400300
Chr03	23186253
Chr04	21661016
Chr05	21501033
Chr06	20216928
Chr07	19623316
Chr08	18957817
Chr09	13499373
ChrX	11379864

Fig. 3.

Chromosomal synteny between Promethis valgipes (Pval) and Tribolium castaneum (Tcas). The X chromosome is highlighted in red.

Table 2.

Genome assembly statistics for Promethis valgipes.

Characteristics	P. valgipes
Genome assembly
Genome Size (Mb)	213.68
Number of scaffolds	19
Number of chromosomes	10
Scaffold N50 length (Mb)	21.50
Contig N50 length (Mb)	17.10
GC (%)	36.97
BUSCO completeness (%)	99.7%
S	99.3%
D	0.4%
F	0.1%
M	0.2%
Mapping ratio (%)
Illumina	90.01%
HIFI	99.99%
RNA-Seq	92.93%

C: complete BUSCOs; S: Complete and single-copy BUSCOs; D: complete and duplicated BUSCOs; F: fragmented BUSCOs; M: missing BUSCOs.

Fig. 2.

Chromosome-scale heatmap showing Hi-C contact frequencies across the Promethis valgipes genome assembly. Blue lines indicate superscaffolds, and both the X- and Y-axes represent cumulative scaffold lengths in megabases (Mb).

Fig. 4.

A circular plot summarizes the genomic landscape of Promethis valgipes. From outer to inner rings, it displays chromosome length (physical scale), GC content, gene density, and the distribution of major repetitive elements, including transposable elements (DNA transposons, SINEs, LINEs, and LTRs) and simple repeats.

Genome annotation

To characterize repetitive elements in the P. valgipes genome, de novo repeat annotation was performed using RepeatModeler v2.0.4³³ with the “-LTRStruct” pipeline to improve LTR retrotransposon detection. A custom repeat library was then constructed by integrating the de novo library with RepBase-20230909³⁴ and Dfam v3.5³⁵. RepeatMasker v4.1.2³⁶ was subsequently used to identify and mask repetitive elements using this combined database. Approximately 89.19 Mb (41.74%) of the genome was identified as repetitive sequences. This composition included 62.07 Mb (29.05%) of unclassified repeats, 15.46 Mb (7.22%) of DNA transposons, 6.48 Mb (3.02%) of long interspersed nuclear elements (LINEs), 3.69 Mb (1.71%) of long terminal repeats (LTRs), 0.066 Mb (0.03%) of short interspersed nuclear elements (SINEs), 0.95 Mb (0.44%) of simple repeats, and 0.58 Mb (0.27%) from other categories (Table 4).

Table 4.

Genome assembly and annotation statistics of Promethis valgipes.

Characteristics	P. valgipes
Genome annotation
Protein-coding genes
Number	13544
Mean gene length (bp)	4,484.4
BUSCO completeness (%)	97.4
Repetitive elements
Size (Mb)	89.19 (41.74%)
DNA transposons (Mb)	15.46 (7.22%)
LINEs (Mb)	6.48 (3.02%)
LTRs (Mb)	3.69 (1.71%)
SINEs (Mb)	0.066 (0.03%)
Unclassified (Mb)	62.07 (29.05%)
Simple repeats (Mb)	0.95 (0.44%)
Other repeats (Mb)	0.58 (0.27%)
ncRNA
Number of ncRNA	456
tRNA	295
rRNA	11
miRNA	60
snRNA	49
Histone3	24
Other ncRNA	17
Function annotation
Number of genes matching Uniprot records	13179
Number of genes with InterProScan annotations	11684
Number of genes with eggNOG annotations	12,424
Number of genes with Enzyme Codes (EC) from eggNOG annotations	3054
Number of genes with COG Functional Categories combining InterProScan and eggNOG results	11688
Number of genes with GO items combining InterProScan and eggNOG results	10501
Number of genes with KEGG pathways items combining InterProScan and eggNOG results	5,189

For non-coding RNA (ncRNA) annotation, Infernal v1.1.2³⁷ was used with the Rfam v14.10³⁸ database, and tRNAs were predicted using tRNAscan-SE v2.0.9³⁹. In total, 456 ncRNAs were identified, including 24 histone, 49 small nuclear RNAs (snRNAs), 60 microRNAs (miRNAs), 295 transfer RNAs (tRNAs), 11 ribosomal RNAs (rRNAs), and 17 other ncRNAs (Table 4).

Protein-coding genes in P. valgipes were annotated using MAKER v3.01.0⁴⁰, integrating homology, ab initio predictions, and transcriptomic alignments to generate high-confidence gene models. Gene-finding models were trained and optimized using combined transcriptomic and protein homology evidence. RNA-seq reads were mapped to the genome using HISAT2 v2.2.1⁴¹, and the alignments were assembled into transcripts with StringTie v2.1.6⁴². Ab initio gene predictions were generated with BRAKER v2.1.6⁴³, integrating GeneMark-ES/ET/EP v4.68_lic⁴⁴ and AUGUSTUS v3.4.0⁴⁵. Models were automatically trained using transcriptome alignments and conserved proteins from OrthoDB v11⁴⁶. Protein homology-based gene prediction was then performed with GeMoMa v1.9⁴⁷, employing protein sequences from five insect species: D. melanogaster⁴⁸ (GCF_000001215.4), A. mellifera⁴⁹ (GCF_003254395.2), B. mori⁵⁰ (GCF_030269925.2), T. molitor⁵¹ (GCF_963966145.1), and T. castaneum¹² (GCF_031307605.1) (Table 5). Gene prediction for the P. valgipes genome identified 13544 protein-coding genes, with an average length of 4484.4 bp. Each gene contained an average of 5.6 exons, 4.5 introns, and 5.5 CDSs, with mean exon, intron, and CDS lengths of 324.3 bp, 660.6 bp, and 293.3 bp, respectively (Table 4). Gene completeness was assessed using BUSCO, which identified 97.4% complete BUSCOs, including 1,331 single-copy and 9 duplicated orthologs, along with 0.4% fragmented (6 genes) and 2.2% missing (30 genes).

Table 5.

Species taxonomic information and accession code of all samples used in this study.

Species	Order	Family	Source
Tribolium castaneum	Coleoptera	Tenebrionidae	NCBI (GCA_031307605.1)
Tenebrio molitor	Coleoptera	Tenebrionidae	NCBI (GCA_963966145.1)
Apis mellifera	Hymenoptera	Apidae	NCBI (GCA_003254395.2)
Bombyx mori	Lepidoptera	Bombycidae	NCBI (GCA_030269925.2)
Drosophila melanogaster	Diptera	Drosophilidae	NCBI (GCA_000001215.4)

Functional annotation of the predicted proteins was performed using multiple complementary approaches. Homology searches against the UniProtKB database were conducted with DIAMOND v2.0.11.1⁵², and Gene Ontology (GO) terms as well as KEGG/Reactome pathway assignments were obtained using eggNOG-mapper v2.0.1⁵³. Protein domain annotation was performed using InterProScan v5.53-87.0⁵⁴, integrating five curated databases: Pfam⁵⁵, SMART⁵⁶, SUPERFAMILY⁵⁷, Gene3D⁵⁸, and CDD⁵⁹. Results from all annotation tools were then integrated to generate a comprehensive functional annotation dataset for the predicted genes of P. valgipes. Functional annotation of P. valgipes identified 11,684 genes with InterProScan annotations and 12,424 genes annotated in eggNOG. In addition, 13,179 genes were matched to UniProt entries, 10,501 were assigned Gene Ontology (GO) terms, 5,189 were mapped to KEGG pathways, 3,054 were associated with Enzyme Commission (EC) numbers, and 11,688 were classified into COG categories. Genome feature distributions, including repeat elements, gene density, and GC content, were visualized across pseudochromosomes using TBtools³².

Data Records

The raw sequencing data and genome assembly of Promethis valgipes are publicly available through the National Center for Biotechnology Information (NCBI). The sequencing datasets, including Hi-C (SRR35493487⁶⁰), transcriptome (SRR35493488⁶¹), Illumina short reads (SRR35493489⁶²), and PacBio HiFi long reads (SRR35493490⁶³), are publicly available under their respective accession numbers. The final genome assembly has been deposited in NCBI under accession number GCA_054205895.1⁶⁴. Genome annotation files, including repeat annotations, gene structure predictions, and functional annotations, are available on Figshare⁶⁵.

Technical Validation

The quality of the P. valgipes genome assembly was evaluated using two complementary approaches. Genome completeness was assessed using BUSCO v5.0.4⁶⁶ in genome mode against the Insecta gene set (insecta_odb10, n = 1,367). The initial Hifiasm assembly achieved a BUSCO completeness of 99.8% (S: 93.9%, D: 5.9%), with only 0.1% fragmented and 0.1% missing BUSCOs. After removal of redundant haplotigs using Purge_Dups, the BUSCO completeness remained at 99.8%, with a substantial reduction in duplicated BUSCOs (S: 99.4%, D: 0.4%), while fragmented and missing BUSCOs accounted for 0.0% and 0.2%, respectively. The marked decrease in duplicated BUSCOs from 5.9% to 0.4% indicates that Purge_Dups effectively removed redundant haplotig sequences while preserving genuine duplicated regions and essential gene content. The k-mer spectrum analyses before and after haplotig removal (Fig. 5a and b) further support the effectiveness of the purging process, demonstrating reduced assembly redundancy without compromising genome completeness. The final chromosome-level assembly achieved a BUSCO completeness of 99.7%, including 99.3% single-copy, 0.4% duplicated, 0.1% fragmented, and 0.2% missing BUSCOs. Assembly accuracy was evaluated using Minimap2 v2.23⁶⁷ and SAMtools v1.22⁶⁸ to map PacBio HiFi, Illumina, and RNA-seq reads to the final assembly. The resulting mapping rates were 99.99%, 90.01%, and 92.93%, respectively. To evaluate the quality of the assembly, Merqury v1.3⁶⁹ was used to identify potential errors through efficient k-mer set operations and QV score calculation. The k-mer completeness of the P. valgipes genome was 96.86%, and the QV score was 45.39. Together, these results confirm a highly complete and accurate genome assembly for P. valgipes.

Fig. 5.

K-mer spectrum analysis of the Promethis valgipes genome assembly before and after haplotig removal. (a) Initial assembly; (b) Purge_Dups-processed assembly.

Ethics statement

This research involved an invertebrate insect species and did not include vertebrate animals. According to the relevant institutional and national regulations, ethical approval is not required for studies involving insects. All procedures were conducted in accordance with accepted ethical standards for invertebrate research.

Author contributions

Jin.J. and Zhu.W. contributed to the research design. Ye.M. and Qiao.J. collected the samples. Jin.J. and Zhu.W. analyzed the data. Jin.J. and Zhu.W. wrote the draft manuscript and revised the manuscript. All co-authors contributed to this manuscript and approved it.

Code availability

No specific script was used in this work. All commands and pipelines used in data processing were executed according to the manual and protocols of the corresponding bioinformatic software.

Data availability

This dataset is original and previously unpublished. The authors declare no competing interests. Raw sequencing reads and the assembled genome for Promethis valgipes are available in the NCBI BioProject database under accession PRJNA1308995. Annotation files, including repeat elements, gene models, and functional predictions, are publicly available on Figshare (https://doi.org/10.6084/m9.figshare.31294198.v3).

Competing interests

The authors declare no competing interests.