Within Cryptorhynchinae subfamily (Coleoptera: Curculionidae), the Neotropical genus Philonis represents an underexplored lineage of stem-galling weevils that are tightly associated with species of Passiflora (Passifloraceae). Among them, Philonis inermis has recently attracted attention as a potential biological control agent against Passiflora foetida, an invasive vine that causes significant ecological and economic damage in Australia (Clavijo-Giraldo et al., 2025. Unpublished). Although a considerable number of cryptorhynchine weevils are known as agricultural pests, some species are being studied for their potential as biological control agents. P. inermis is one such example, exhibiting high host specificity towards P. foetida and inducing gall formation that weakens or kills the host plant. However, the lack of molecular data for Philonis hampers its accurate phylogenetic placement and limits the development of molecular tools for its identification and monitoring in both native and introduced ranges.

Mitochondrial genomes or mitogenomes have become fundamental tools for understanding the evolutionary history, systematics, and ecology of insects. ^{1, 2} In Coleoptera, the increasing availability of complete mitogenomes has facilitated robust phylogenetic analyses at various taxonomic levels, revealing patterns of diversification and adaptation in highly speciose families such as Curculionidae and other related weevil lineages. ^{3– 5} Despite these advances, mitogenomic data remain scarce for the subfamily Cryptorhynchinae, which encompasses a highly diverse assemblage of weevils with complex host-plant interactions.

Although mitogenomes are powerful markers, their use in phylogenetic studies is not without limitations. ^{6, 7} High substitution rates—particularly in third codon positions—can lead to substitution saturation in deep evolutionary branches, reducing the ability to accurately recover relationships among distantly related taxa. Such constraints are less problematic at shallow timescales, where mitochondrial genomes retain strong resolving power for population-level analyses and recent divergences. Because the present study focuses on intraspecific and closely related lineages, mitogenomic data remain well suited to our research objectives. ⁸

Recent mitogenomic studies within Cryptorhynchinae have mainly focused on species of economic concern, particularly pests of woody plants and crops. Examples include Eucryptorhynchus chinensis and E. brandti, pests of Ailanthus altissima in China, ^{9, 10} and the mango seed weevils Sternochetus gravis, S. mangiferae, and S. olivieri. ¹¹ The hyperdiverse genus Trigonopterus, with hundreds of recently described species, ¹² also provides important comparative resources. Although related genera such as Aclees ¹³ are not part of Cryptorhynchinae, they remain relevant for broader curculionid comparisons. In parallel with mitogenomic research, the use of the mitochondrial Cytochrome c oxidase subunit I (COI) gene as a DNA barcode has become an essential tool for species identification, biodiversity assessment, and the detection of cryptic diversity in weevils and other insects. ^{14, 15} Despite its broad application in Curculionidae, COI barcoding data have been scarce for Neotropical Cryptorhynchinae, limiting our understanding of species boundaries and population structure in this group. Until now, no complete mitogenomes have been available for any Neotropical representative of Cryptorhynchinae, making P. inermis the first of its kind. The integration of mitogenomic and COI barcode data presented here provides a valuable reference for future studies on species delimitation, comparative genomics, and the development of molecular tools for monitoring and management of potential biological control agents.

In this context, the accurate delimitation of P. inermis is critical for any potential classical biological control strategy against P. foetida. Correct species identification, coupled with low intraspecific genetic variability, ensures the reliability and safety of introducing candidate agents into new environments. By combining complete mitochondrial genome sequencing with COI barcode analysis, this study not only provides the first comprehensive molecular characterization of P. inermis, but also demonstrates the utility of COI as a diagnostic marker for its unambiguous identification. These resources will serve as a foundation for both fundamental studies on Cryptorhynchinae evolution and applied research aimed at evaluating P. inermis as a safe and effective biocontrol agent.

This study aims to generate the first low-coverage genome sequencing–based mitochondrial genome of Philonis inermis and to integrate these data with COI barcode sequences in order to refine molecular identification and enable comparative analyses within Cryptorhynchinae. Through comparative analyses with available mitogenomes of related genera ( Eucryptorhynchus, Sternochetus, and Trigonopterus), we explore patterns of gene arrangement, nucleotide composition, codon usage, and control region variability. These results not only fill a significant gap in the mitogenomic data of Neotropical Cryptorhynchinae but also provide essential molecular resources for evaluating P. inermis as a candidate biological control agent of P. foetida, linking fundamental evolutionary insights with applied biocontrol strategies.

Despite family Curculionoidae is rendering as one of the most diverse groups of Coleoptera, encompasses approximately 62,000 species distributed among 5,800 described genera, ²⁸ many attributes about genome structure, diversity, and ecological role of Neotropical species belonging to Crypthorynchinae subfamily such P. inermis is poorly unknowledge so far.

The weevils of the Neotropical genus Philonis (Curculionidae: Cryptorhynchinae) represent a largely unexplored lineage within the diverse assemblage of gall-inducing insects. Here, we present the first genomic resources for this genus based on low-coverage genome sequencing, including the complete mitochondrial genome and an assessment of COI DNA barcode accuracy. These data provide a foundation for future studies of genetic diversity, population structure, and evolutionary history, and may also inform applied research in biological control of the invasive vine Passiflora foetida L.

The relatively fragmented assemblies reflect the shallow genome-skimming strategy adopted in this study, which was specifically designed to recover the complete mitochondrial genome and representative nuclear markers rather than to generate a high-contiguity nuclear assembly. Our primary objective was to establish genomic resources for taxonomic validation and phylogenetic inference, for which modest sequencing depth is sufficient. Although increased coverage and the incorporation of long-read technologies would substantially improve assembly contiguity, such approaches were beyond the scope of the present study.

Consistent with the limited coverage, BUSCO profiles indicate that neither assembly captures the complete nuclear gene space; nonetheless, we recovered 196 shared single-copy orthologs, 191 of which contain intact open reading frames and were assigned to 22 COG functional categories. These loci encompass core informational and metabolic functions and collectively constitute a robust marker panel for future comparative and phylogenomic analyses in Philonis inermis and related Neotropical Cryptorhynchinae. The substantial proportion of unclassified COGs further highlights the limited genomic characterization of this lineage and underscores the potential for discovery as additional genomic data become available. In parallel, the recovery of duplicated BUSCOs and extensive rRNA and histone gene clusters provides complementary multicopy markers that may prove useful for resolving deeper or more complex evolutionary relationships. Future work integrating long-read sequencing and Hi-C scaffolding will be essential to resolve repetitive regions, reduce fragmentation, and ultimately achieve chromosome-scale assemblies for this ecologically important genus.

The mitochondrial genome of P. inermis exhibits a nucleotide composition biased toward (A+T), as well as gene order and orientation that are highly conserved, and consistent with the ancestral insect mitogenome architecture. Comparative analyses with complete mitogenomes from other Cryptorhynchinae genera revealed strong structural and syntenic conservation across the group. ^{9, 10, 12, 29} As reported for other species such as Eucryptorrhynchus chinensis and E. brandti the mitogenome of P. inermis lacks an identifiable deficiency of tRNA-Ile gene. ¹⁰ The trnI gene is likely located within the highly variable control region, where high elevated divergence hampers automated annotation and precise boundary delimitation, making its detection a common challenge. ³⁰

Mitogenome-based tree place Philonis close to Eucryptorrhynchus, but we treat this as preliminary, given the limited and uneven mitogenomic sampling across Cryptorhynchinae. Broad, multi-gene frameworks show that relationships above the genus level can be difficult to stabilize in this subfamily, and that extensive sampling across loci is required to resolve deeper nodes. ^{31, 32} In particular, Riedel et al. ³² recovered a large-scale molecular phylogeny that points to an American origin for Cryptorhynchinae and highlights the value of integrating mitochondrial and nuclear markers for robust placement. Our results align with this perspective: the topology we report is a useful working hypothesis that should be tested with expanded taxon and locus coverage.

If proximity to Eucryptorrhynchus is confirmed, the comparison is ecologically instructive. Ailanthus altissima (Simaroubaceae), the tree-of-heaven, is a fast-growing invasive tree native to China that readily colonizes disturbed habitats and can displace native vegetation. Within this host context, E. scrobiculatus and E. brandti are highly specialized on A. altissima. In parts of their native range, they are considered pests; however, they have also been evaluated as potential biological control agents where A. altissima is invasive, and climate-suitability assessments suggest differential responses of the two congeners to future climates. ^{10, 33} This dual “pest vs. biocontrol” context underscores why clear systematics and robust diagnostics matter when considering host-specific herbivores for applied programs.

Interestingly, all other cryptorhynchine weevils with complete mitochondrial genomes currently reported are documented agricultural pests of economic importance in Asia or Oceania, highlighting a significant gap for Neotropical species. In contrast, P. inermis is a stem-galling specialist with a narrow host range restricted to P. foetida, an invasive vine in Australia. ³⁴

At a broader scale, historical biogeography indicates a complex macroevolutionary backdrop for Cryptorhynchinae, with major diversity centers in the Neotropics and Australasia and signals consistent with an American origin. ^{3, 32, 35} Comparative insights from flightless Trigonopterus show repeated crossings of major biogeographic barriers (e.g., Wallace’s Line), rapid radiations and finely structured endemism, and these features complicate deep-time reconstructions when sampling is sparse. ³⁶ These patterns provide a cautionary frame for interpreting single-marker or low-taxon trees and reinforce the need for expanded, balanced sampling when refining the placement of Philonis.

Assessment for COI gene DNA barcoding regions indicates high accuracy on molecular identification of P. inermis, with minimal genetic divergence among Colombian individuals from three populations (K2P distances ≤ 0.006). This low intraspecific variability underscores the genetic coherence of the species and confirms that the sampled populations across Colombia belong to the same taxonomic unit. Despite the limited flight ability of P. inermis, this genetic homogeneity may be influenced by passive dispersal associated with its host ( P. foetida), a hypothesis that warrants further investigation. Furthermore, both inter – and intraspecific genetic distance gaps support the COI barcode as a suitable tool for species identification in Neotropical cryptorhynchine weevils, exceeding values reported for related species from Asia, Europe, and Oceania. ^{36, 37} Under this scenario, our COI results supports unambiguous diagnosis of P. inermis. This pattern aligns with the central rationale for DNA barcoding as a standardized, taxonomically integrative tool. ^{15, 38} The maximum likelihood tree constructed from COI sequences clustered all P. inermis specimens together with strong statistical support, further confirming its genetic uniformity and separation from other Neotropical taxa. Collectively, these barcode results provide a robust molecular baseline for species identification and support the consideration of P. inermis as a promising candidate for biological control programs targeting P. foetida.

From an applied perspective, the combination the mitogenome and COI barcoding supports accurate recognition across Colombian populations, which is a prerequisite for any subsequent risk assessment. Furthermore, this approach strengthens integrative taxonomy, biosecurity, and classical weed biocontrol workflows. Barcodes offer interoperable, scalable diagnostics for look-alike taxa and immature stages, facilitate data sharing across laboratories and jurisdictions, and support post-release monitoring, both of which are key steps to minimize non-target risk. ^{15, 38, 39} The global invasive potential documented for S. mangiferae under climate change further illustrates why strong diagnostics and early detection pipelines are increasingly critical for curculionid lineages with agricultural relevance. ⁴⁰

Limitations and next steps follow directly from our results. First, phylogenetic inferences from current mitogenome sampling should be treated as provisional. Resolving deeper nodes will require denser Neotropical sampling, including close relatives of Philonis, and multi-locus or genomic matrices that integrate nuclear markers. Second, expanding the geographic and host-associated sampling for COI (and additional markers) will help quantify population structure and confirm the breadth of the barcode gap. Third, low-coverage, short-read genome skimming inherently underrepresents repetitive and GC-extreme regions and can collapse recent paralogs, yielding fragmented assemblies and biasing functional annotations toward conserved single-copy genes; increasing sequencing depth and integrating long reads and Hi-C will mitigate these biases. Together, these steps will strengthen both the systematic placement of Philonis and the applied utility of its genetic resources for monitoring and potential biocontrol assessment.

We provide the first mitogenomic reference for P. inermis (15,120 bp; ~77% A+T), with conserved gene order and deficiency of tRNA-Ile likely obscured within the variable control region. Maximum-likelihood analyses recover P. inermis as a well-supported clade and tentatively near Eucryptorrhynchus, a hypothesis that awaits denser taxon and locus sampling. COI barcodes from 20 Colombian individuals show extremely low intraspecific divergence (K2P ≤ 0.006) and a pronounced barcode gap from other American cryptorhynchine weevils, enabling reliable, field-ready diagnostics. Low-coverage genome sequencing recovered 196 single-copy orthologs along with a complementary set of duplicated BUSCOs and multicopy rRNA and histone genes, furnishing anchors for future genome-scale phylogenetics and comparative genomics. Together, these resources demonstrate the utility of COI barcoding for the current molecular identification of P. inermis, establish a molecular foundation for systematics and population-level studies, and inform the risk-aware evaluation of P. inermis as a host-specific candidate for classical biological control of P. foetida. Priority next steps include long-read and Hi-C genome assemblies and expanded geographic and taxonomic sampling—including nuclear markers—to resolve deeper relationships and better quantify population structure.