Habitat loss is causing declines in native bees and reducing pollination services. Revegetation can be used to reverse these declines, and this restoration technique attracts growing efforts and resources. However, how the quality of revegetation affects native bee abundance, diversity and their pollination services is not well understood, and this limits opportunities to improve revegetation outcomes. To assess this gap, we surveyed floral and bee diversity in revegetated landscapes ranging in habitat quality, and compared these among each other and to remnant habitat and cleared areas. We also measured pollination services using two native phytometer species, which can be pollinated by native bees only, or by both native and introduced honey bees (Apis mellifera). We found that bee diversity and richness were higher within treatments that were higher in floral diversity. In addition, while pollination services provided by honey bees were uniform across treatments, remnant vegetation supported greater pollination services to the plant species pollinated by native bees only. These results indicate that higher quality revegetation characterized by the establishment of a more diverse set of plant species, has the potential to restore native bee diversity and associated pollination services. However, for the plant species investigated, restoration of pollination services had not occurred and might require more time. These results suggest preserving remnant vegetation should be the highest priority conservation action, and that restoration practitioners and landowners wishing to support landscape‐level bee diversity and pollination services, should aim for revegetation using high flowering plant diversity.

Mussels exhibit a sophisticated innate immune response characterized by many highly variable molecules responsible for recognizing and killing potential pathogenic microorganisms. The complexity of this molecular arsenal is marked by the occurrence of gene presence-absence (PAV), a phenomenon that targets numerous expanded lineage-specific gene families. This phenomenon enhances inter-individual sequence variability, further enriching the diversity of the repertoire of molecules involved in the immune response. Until now, the origin of mussel dispensable genes, which, unlike core genes, are not shared by all individuals, has remained elusive. In this study, by analyzing the resequenced genomes of more than 160 individuals in four distinct species of the Mytilus complex, we characterize the repertoire of mytilin genes encoding hemocyte-specific antimicrobial peptides (AMPs). We define a canonical gene architecture comprising four protein-coding genes and two pseudogenes in most haplotypes. However, the organization of the locus displays a marked intra-specific diversity due to the presence of variable alleles, the frequent pseudogenization of mytilin G1 and structural variants associated with additional dispensable mytilin genes, which often retain features that support functional preservation. Molecular phylogeny supports an ancient origin for dispensable mytilin genes, predating the radiation of modern Mytilus species. This suggests that most widespread extant haplotypes derive from a larger and more complex ancestral mytilin gene cluster and that dispensable mytilin genes are vestigial AMPs that have been retained only in a few populations where their presence may have contributed to fitness advantages and local adaptation.

Pollen identification (ID) and quantification is important in many fields, including pollination ecology and agricultural sciences, and efforts to explore optimal molecular methods for identifying low concentrations of DNA from plant mixtures are increasing, but quantifying mixture proportions remains challenging. Traditional pollen ID using microscopy is time‐consuming, requires expertise and has limited accuracy and throughput. Molecular barcoding approaches being explored offer improved accuracy and throughput. The common approach, amplicon sequencing, employs PCR amplification to isolate DNA barcodes, but introduces significant bias, impairing downstream quantification. We apply a novel molecular hybrid capture approach to artificial pollen mixtures to improve upon current taxon ID and quantification methods. The method randomly fragments DNA and uses RNA baits to capture DNA barcodes, which allows for PCR duplicate removal, reducing downstream quantification bias. Four reference databases were used to explore identification and quantification. A restricted matK database containing only mixture species yielded sequence proportions highly correlated with input pollen proportions, demonstrating the potential usefulness of hybrid capture for metabarcoding and quantifying pollen mixtures. Identification power was further tested using two reference libraries constructed from publicly available sequences: the matK plastid barcode and RefSeq complete chloroplast references. Single barcode‐based taxon ID did not consistently resolve to species or genus level. The RefSeq chloroplast database performed better qualitatively but had limited taxon coverage (relative to species used here) and introduced ID issues. At the family level, both databases yielded comparable qualitative results, but the RefSeq database performed better quantitatively. Whilst the method developed here has tremendous potential, the choice and expansion of reference databases remains one of the most important factors allowing qualitative and quantitative accuracy using the full set of genomic regions screened by this hybrid capture method.

Insects are commonly expected to be protected from genomic erosion by high fecundity, short generation times, and large census sizes. Yet, insect populations can decline and eventually go extinct, underscoring the need for genomic indicators that provide actionable early warnings of population collapse. Here, we test this expectation by comparing contemporary and historical genomes of the Ponza grayling, Hipparchia sbordonii , an endangered butterfly endemic to the Pontine Islands in the Mediterranean, with the genomes of a widespread European congeneric species, H. semele . Using whole-genome resequencing, outgroup-based variant polarization, demographic reconstruction, runs of homozygosity, selection scans, and annotation-based genetic-load analyses, we show that H. sbordonii has undergone sustained demographic contraction, including a sharp recent decline. Despite limited temporal change in mean genome-wide heterozygosity, we observe extensive autozygosity, elevated inbreeding, and a clear shift from masked to realized genetic load in contemporary H. sbordonii . R’ _XY analysis revealed similar relative frequencies of high-impact derived variants in H. sbordonii and H. semele , suggesting ineffective purging during population collapse, whereas low- and moderate-impact variants were relatively enriched in H. sbordonii , particularly within candidate regions under selection. This indicates a more complex dynamic, in which functional variation has been shaped by the combined effects of drift, relaxed purifying selection, and possible local adaptation. Our study shows that declining butterfly populations bear distinctive signatures of genomic erosion, mirroring patterns well documented in vertebrates. Yet recent demographic collapse in H. sbordonii is more clearly captured by long runs of homozygosity and realized genetic load than by changes in mean genome-wide heterozygosity, highlighting their potential as early warning indicators for monitoring declining insect populations.

Background: The overwhelming majority of the antimicrobial peptides (AMPs) studied in mussels (Mytilus spp.) so far are specifically expressed by hemocytes and display compact disulfide-stabilized structures. However, gill-specific myticalins play a role in mucosal immunity and are one of the very few examples of known molluscan AMPs lacking cysteine residues. Methods: We investigate the molecular evolution of myticalins, compiling a collection of sequences obtained by carefully annotating 169 genome assemblies of different Mytilus species. We determine the gene presence/absence patterns and gene expression profiles for the five myticalin subfamilies, including the newly reported myticalin E. Results: All sequences are deposited in MyticalinDB, a novel database that includes a total of 100 unique mature myticalin peptides encoded by 215 protein precursors, greatly enriching the compendium of these molecules from previous reports. Among the five subfamilies, myticalin A and C are the most widespread and highly expressed across all Mytilus species. Interestingly, structural prediction reveals a previously unreported strong amphipathic nature for some myticalins, which may be highly relevant for their biological activity. Conclusions: The results reported in this work support the role of myticalins in gill-associated mucosal immunity and highlight the importance of inter-individual molecular diversity in establishing an efficient response to microbial infections. The newly established MyticalinDB provides a valuable resource for investigating the evolution and extraordinary molecular diversity of this AMP family.