**Background:** Bark beetles (Scolytinae) are subcortical herbivore-fungivores that often form symbiotic relationships with fungi, particularly ophiostomatoid fungi, which can aid in overcoming host defenses or improving larval nutrition. While conifer-killing bark beetles are well-studied, few hardwood-killing bark beetles exist, and their fungal associations remain poorly understood. The alder bark beetle, Alniphagus aspericollis, colonizes and kills red alder (Alnus rubra), a nitrogen-fixing hardwood in the Pacific Northwest. This study aimed to identify filamentous fungal associates of A. aspericollis and characterize the consistency of beetle–fungus relationships, testing the prediction that A. aspericollis would not be consistently associated with nitrogen-provisioning ophiostomatoid fungi due to the nitrogen-rich tissues of its host.
**Methods:** Adult A. aspericollis and gallery phloem samples were collected from seven sites across the Greater Vancouver region, British Columbia, Canada, between 14 July and 26 August 2015. One to three infested red alder hosts were sampled per site, with three adult beetles extracted from separate entrance holes and non-overlapping galleries per tree (total n = 54 beetles). A 5 × 5 cm phloem section was also taken from each gallery. Fungal isolations were performed within one week: beetles were washed with sterile water and the inoculum spread on malt extract agar (MEA); phloem chips were surface-sterilized and plated on water agar. Filamentous fungi were sub-cultured to purity and identified by DNA barcoding of the internal transcribed spacer (ITS) region, with additional gene regions (RPB2, EF1-α, beta-tubulin) sequenced for the most dominant isolates. Phylogenetic analyses using maximum likelihood (PhyML 3.0, HKY85 substitution model) were conducted for Neonectria, Ophiostoma, and Cadophora isolates.
**Key Results:** Fungi were identified to genus or species level for 89% of cultures (≥99% sequence homology). The most frequently isolated fungus was a previously undescribed Neonectria major-like species (Neonectria sp. nov.), isolated from 66.7% of adult beetles (36/54), 59.3% of gallery phloem samples (32/54), and 94.4% of beetle-infested trees (17/18). Concurrent isolation from both beetle and phloem from the same gallery occurred in 53.7% of samples (29/54). Phylogenetic analyses of ITS, RPB2, and EF1-α sequences placed Neonectria sp. nov. in a well-supported distinct clade (88–98% bootstrap support) most closely related to N. major and N. ditissima. Ophiostoma quercus was the second most frequent associate, isolated from 27.7% of adult beetles (15/54), 9.3% of phloem samples (5/54), and 55.6% of trees (10/18), but concurrent isolation from both sources occurred in only 5.6% of galleries (3/54). A putatively novel Ophiostoma sp. was isolated from 5.6% of beetles (3/54) and 3.7% of phloem samples (2/54), exclusively at Burnaby Mountain. Cadophora spadicis, a new record for red alder, was isolated from 11.1% of beetles (6/54) and 3.7% of phloem samples (2/54), with no concurrent isolations. Other fungi (Penicillium spp., Beauveria spp., Cladosporium sp., etc.) were rarely isolated. Ophiostomatoid fungi were only loosely associated with A. aspericollis, contrasting with the consistent associations seen in conifer-killing bark beetles.
**Clinical Implications:** This study identifies a novel Neonectria species as a consistent associate of A. aspericollis, suggesting a potential vector relationship where the beetle may transmit this fungus between red alder trees. The loose association with ophiostomatoid fungi supports the hypothesis that nitrogen-fixing hosts reduce selection for nitrogen-provisioning fungal symbionts. The findings expand understanding of hardwood-killing bark beetle ecology and highlight a previously unknown beetle–fungus system. Future research should clarify whether the Neonectria sp. nov.–A. aspericollis relationship is mutualistic, commensal, or antagonistic, and assess the pathogenicity of Neonectria sp. nov. and the novel Ophiostoma species.